Filamentous fungal biomats, methods of their production and methods of their use

ABSTRACT

A novel method of growing fungi is disclosed which uses an engineered artificial media and produces high density filamentous fungi biomats that can be harvested with a minimum of processing and from which fungal products such as antibiotics, proteins, and lipids can be isolated, the method resulting in lowered fungus cultivation costs for energy usage, oxygenation, water usage and waste stream production.

This application is a Continuation Application of co-pending PCTApplication No. PCT/US17/20050, filed Feb. 28, 2017, which claimspriority under 35 U.S.C. 119(e) on U.S. Provisional Application No.62/302,123, filed on Mar. 1, 2016, U.S. Provisional Application No.62/340,381, filed on May 23, 2016, and U.S. Provisional Application No.62/345,973 filed on Jun. 6, 2016, the contents of each of which ishereby incorporated by reference in its entirety for all purposes.

TECHNICAL FIELD

This application relates to isolated filamentous fungal strains withinthe Ascomycota, Zygomycota, Basidiomycota, Glomermycota, andChytridiomycota phyla, such as Fusarium species, Aspergillus species,Tricoderma species, Penicillium species, species within the Mucorales,including Rhizopus species, the acidophilic filamentous fungal straindesignated as MK7 and their progeny as well as methods of conductingsurface fermentation to produce filamentous fungi biomats from suchfungal strains which produce a large variety of useful products.

BACKGROUND

The cells of most fungi grow as tubular, elongated and thread likestructures called hyphae which may contain multiple nuclei and whichextend by growing at their tips. This is in contrast to similar-lookingorganisms, such as filamentous green algae, which grow by repeated celldivision with a chain of cells.

The collective body of hyphae that constitutes the vegetative stage of afungus is called a mycelium (plural mycelia). The mycelium can beconsidered the main body or form of the fungus and is often described as being filamentous. Growth occurs by the asexual reproduction of hypha,which grow into branching chains. Mycelium is important to the fungusbecause it can navigate through soil or wood and use that substrate asfood, which the fungus will need if it is to produce fruit bodies (e.g.,basidoiocarps) such as mushrooms, brackets, truffles, cups, or morels.

Mycelia excrete exoenzymes which can kill living tissue (necrotrophic)and then absorb that dead material (saprotrophic), simply absorbingmaterial that was already dead (again, saprotrophic), or by feeding offof living tissue (biotrophic).

While it is believed that all of the phyla of the Fungi kingdom containfilamentous species, the Ascomycota and Zygomycota phyla, in particularhave a large number of filamentous species. The members of these phylamake a large variety of products such as proteins, amino acids, oils,medicinals (e.g., penicillin), food (e.g., tempeh), food additives, foodpreservatives (e.g., citric acid), and industrial enzymes as well asbeing used in baking, and the production of chees, beer, and wine.

State of the art solid-substrate fermentation (SSF) suffers from anumber of distinct disadvantages. For example, the final product, i.e.the produced biomass, is intimately mixed with the solid substrate whichis fundamentally difficult to separate one from the other. Typically,SSF produces fungal biomass in low concentrations, has very lowconversion rates and ultimately results in low yields. SSF requiresspecific water activities for effective fermentation. Delivering andmaintaining the right amount of water activity is difficult andexpensive to implement. Aerating SSF systems is also difficult toaccomplish, further exacerbating conversion efficiencies and limitingsystem yields. Improper water activity as well as poor aeration poselimitations to mass and heat transfer, which result in overheating anddeficiencies in oxygen supply. The resulting biomass is characterized ashaving randomly oriented filaments, which greatly limit utility incertain applications; i.e. food and/or animal feed.

Quorn™, a product comprised primarily of the biomass of Fusariumvenenatum filamentous fungi offers a relatively nutritional mycoprotein.Quorn™ is produced by a state of the art submerged fermentation system,capable of producing large volumes in a batch based continuous process.Although commercially viable, the production methodology suffers from anumber of distinct disadvantages. In order to meet commercial demands,Quorn™ uses bioreactors that cost between $35-40 million each. TheQuorn™ system is run continuously in a single reactor until the fungalsystem matures beyond key metrics or is contaminated by another species.At this point, production comes to a standstill, the reactor and allassociated plumbing is emptied and sterilized, a process that can takeweeks to complete and introduces a number of serious issues for asupplier of commercial product. Such issues are, for example, (1)difficult to predict production cycles, (2) costs incurred for cleaningand stopping production, (3) difficulties in controlling inventory, etc.Further, submerged fermentation in large bioreactors requires tremendousamounts of energy to aerate and mix. Separation of the biomass from theliquid in which it ferments requires centrifugation, which is also knownto be a capital intensive and energy demanding process. The process isfurther water intensive, necessitating the handling of large amounts ofwaste water. The biomass produced is characterized as having shortfilament lengths, which limits its ability to directly convert tofood/feed products without introducing bind agents and subsequentprocess steps which incur further costs, difficulties and effort toeffectively manage.

The present filamentous mycelia growth methodologies suffer from anumber of disadvantages. For example, facilities having the properaeration and equipment needed for fungal growth and subsequentseparation of the fungal mycelia from the growth media (e.g.,centrifuges) require significant capital expenses, especially forconducting fungal growth on an industrial scale. Not only do the currentprocesses require substantial energy and water inputs, but they alsoresult in the generation of a large waste stream.

Consequently, there is a need in the industry for a streamlined approachto filamentous mycelia filamentous fungi biomat formation.

SUMMARY OF THE INVENTION

The current disclosure overcomes the limitation of the processescurrently used. Here, filamentous fungi biomats are generated viasurface fermentation after inoculation of the desired fungal strain intoa novel growth media where no aeration is required. This method ofsurface fermentation is applicable to a large variety of fungal specieswhich are able to produce a wide assortment of products across aspectrum of different industries. The media developed generates rapidcell growth, creates high density filamentous fungi biomats with longfilaments, produces small waste streams, and allows engineering of thefilamentous fungi biomat produced as a function of carbon source, carbonto nitrogen ratio (C:N), and process parameters. The overall effect isone in which high production rates occur with minimal environmentalimpact as measured by water usage, energy usage, equipment requirements,and carbon footprint.

Therefore, the current disclosure provides an artificial media suitablefor culturing filamentous fungi and enabling their production of afilamentous fungal biomat. The artificial media comprises at least thefollowing macronutrients: nitrogen (N), phosphorus (P), calcium (Ca),magnesium (Mg), carbon (C), potassium (K), sulfur (S), oxygen (O),hydrogen (H) and the following trace nutrients: iron (Fe), boron (B),copper (Cu), Manganese (Mn), molybdenum (Mo), and zinc (Zn). In someinstances, the trace nutrients are augmented with the followingadditional trace nutrients: chromium (Cr), selenium (Se), and vanadium(V). The artificial media has varying C:N ratios which favor productionof filamentous fungi biomats having either a high protein:lipid ratio ora high lipid:protein ratio.

Also provided are conditions culturing various filamentous fungi forfilamentous fungi biomat production, some of which are acidophilic, suchas species and/or strains of Fusarium, Fusisporium, Pseudofusarium,Gibberella, Sporotrichella, Aspergillus. Penicillium, Triocoderma,species within the Mucorales sp. (e.g., Rhizopus sp.) and thefilamentous fungal strain designated as MK7. Depending on the speciesand/or strain, the pH of the culturing media ranges from about 0.68 toabout 8.5 and in some cases up to 10.5. One embodiment of the methodincludes inoculating one or more of the fungal species and/or strain(s)into artificial media and growing the fungal species and/or strain(s) toproduce filamentous biomass which contains one or more useful products.

The filamentous fungi biomats produced arise from anaerobic,microaerobic, aerobic conditions of a combination thereof via surfacefermentation. The filamentous fungi biomats comprise the fungal speciesand/or strain and/or progeny thereof in the form of conidia,microconidia, macroconidia, pycnidia, chlamydospores, hyphae, fragmentsof hyphae, or any and all combination thereof.

Also provided are methods for harvesting the filamentous fungi biomats,isolation and/or purification of useful proteins, amino acids, and/orlipids produced by the filamentous fungi. These proteins, amino acids,and/or lipids can be used in food, fish feed, animal feed, oils, fattyacids, medicinals, nutraceuticals, fungicides, herbicides, yeasticides,insecticides, biolubricants, and as a feedstock for conversion to othervalue added products.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. A, B: strain MK7 in nature in a hot spring environment atYellowstone National Park; C, D: strain MK7 biomass produced underdistinct artificial conditions showing high density, high tensilestrength cohesive pure biomass; E: cross section of strain MK7 biomassof C, D.

FIG. 2. Exemplary 10 L bioreactors used for the generation of inoculum.

FIG. 3A: Loop showing white mycelial mat collected from 2^(nd)generation archive culture; 3B: Petri plate with strain MK7 mycelialmat.

FIG. 4. Growth and pH of strain MK7 culture in 10 L bioreactor to beused for inoculation of tray reactors. MK7-1 liquid medium at 7.5%glycerol content with a C:N ratio of 7.5:1. Optimal culture for using asinoculum is generated between 72 and 90 hours when the biomass is in thelate exponential growth phase (between arrows).

FIG. 5A: Trays used a s tray reactors for producing biomat. The ruler inthe tray is 31.75 cm (12.5 inches) long; FIG. 5B: Bioreactor consistingof a tray rack system used to hold 39 plastic trays. The whole reactoris wrapped in Saran®-like clear plastic wrap.

FIG. 6. Harvested strain MK 7 biomat cultivated for high-lipidproduction under limited nitrogen conditions (C:N ratio of 40:1) after 8days of surface fermentation in a 0.25 m² tray with 1.5 liters of MK7-1medium and 125 g/L glycerol.

FIG. 7. Typical growth pattern for strain MK7 in shallow trays showinglag phase, where biomass accumulation rates are relatively slow 0-1.5days), and time of biomat formation (arrow, 1.5 days) when exponentialgrowth begins. Biomat grown in MK7-1 medium with 7.5% glycerol and 30:1C:N ratio.

FIG. 8. Dry weights of strain MK7 biomats grown on glycerol in traysizes ranging three orders of magnitude.

FIG. 9. Linolenic acid production by strain MK7 as a function ofcultivation duration and temperature. 4% glycerol surface fermentation;pH 2.8 and MK7-1 medium.

FIGS. 10A-10C. Transmitted light microscopic images of cross sections of5 days old MK7 biomats produced using MK7-1 medium+glycerol. FIGS. 10A,10B: 50× zoom showing three layers: aerial hyphae layer, transition zonelayer, and dense bottom layer; FIG. 10C: 50× zoom showing two distinctlayers.

FIGS. 11A-D. Cross sectional micrographs of 5 days old MK7 biomatproduced using MK7-Urea medium. FIG. 11A: Top surface of strain MK7biomat revealing aerial hyphae and mycelia extending out from densemycelial layer. Image generated using transmitted light microscope at100× magnification; FIG. 11B: Top surface of strain MK7 biomat revealingaerial hyphae and mycelia extending out from dense mycelial layer. Imagegenerated using transmitted light microscope at 400× magnification; FIG.11C: Bottom surface of strain MK7 biomat revealing hyphae and mycelia.Image generated using transmitted light microscope at 400×magnification; FIG. 11D: Dense interior of strain MK7 biomat revealingits intertwined fibrous composition. Image generated using transmittedlight microscope at 400× magnification.

FIG. 12. A, B: Biomat of Rhizopus oligosporus grown on 0.25 m² tray for6 days using MK-7 medium at pH 4.1 with 5% glycerol. C: 400× lightmicroscope image of Rhizopus oligosporus hyphae in mat.

FIG. 13. Images of Fusarium venenatum grown in 0.25 m² tray reactorafter A: 4 days, and B: 6 days. The biomats were grown using MK7-1medium at pH 5.0 and 12.5% glycerol. Image C shows the hyphal form of F.venenatum taken a 400× magnification using a light microscope. Underthese conditions, F. venenatum produced an average of 71 g of drybiomass per tray for two trays.

FIG. 14. A: Harvesting strain MK7 biomass cultivated via solid-statefermentation (SSF) showing strain MK7 completely integrated inlignocellulose at <5 g strain MK7 dry weight biomass/L (media: feedstockmixture). B: Midrograph image of harvested strain MK7 biomass of Ashowing filaments randomly integrated with wheat straw. C: strain MK7biomat by solid substrate surface fermentation (SSSF) showing dense (180g·L), cohesive essentially pure strain MK7 biomass.

FIG. 15. Cultivation of strain MK7 with various treatments in 12.7×12.7cm trays for 7 days. Error bars are standard deviations of three trays.

FIG. 16. Left: optical microscopic image of strain MK7 cultured with12.5% glycerol at pH 2.7 after 8 days. Right: fluorescent image afterNile Red staining indicating a high percentage of lipid estimated atbetween 40-60% of cell area.

FIG. 17. Lipid profiles produced by strain MK7. (Left Panel) Average oftotal fatty acid methyl esters (FAME) in direct transesterification(total fuel potential) and extractable lipid fractions as a function ofmedia C:N ratio (n=3). Bars within the extractable lipid fraction barrepresent tri-, di- and mono-acyl glycerides (TAG, DAG, MAG) and freefatty acids (FFA) components. Inset shows a GC-FID chromatogram with TAGmolecules dominating the lipid fraction. (Right Panel) FAME profile oflipids generated from direct transesterification of all fatty acids(Direct) to FAME, and FAME derived from only extractable lipidprecursors (Extractable). Inset shows GC-MS chromatograms for the Directand Extractable fractions.

FIG. 18. Strain MK7 biomats after 7 days of growth on Acid WheySurrogate mediums (AWS) at an initial pH of 4.8 (A, B, C). Transmittedlight microscope image (400×) of biomat (C) showing filamentous natureof the material.

DETAILED DESCRIPTION Definitions

As used herein, the verb “comprises,” and its conjugations are used inthis description and in the claims, in its non-limiting sense to meanthat items following the word are included, but items not specificallymentioned are not excluded. In addition, reference to an element by theindefinite article “a” or “an” does not exclude the possibility thatmore than one of the elements are present, unless the context clearlyrequires that there is one and only one of the elements. The indefinitearticle “a” or “an” thus usually means “at least one.”

As used herein, the term “derived from” refers to the origin of source,and may include naturally occurring, recombinant, unpurified, orpurified molecules. A fungus derived from a specific, isolated fungalstrain and/or its progeny may comprise certain mutations but stillretain one, two or more, or all of the distinguishing morphological andphysiological characteristics of the isolated fungi or its progeny fromwhich it was derived.

As used herein, the term “acidophilic” refers to an organism whoseoptimal growth conditions are under acidic conditions.

As used herein, the term “feedstock” refers to any renewable, biologicalmaterial that can be used directly as a fuel, or converted to anotherform of fuel or energy product. Biomass feedstocks are the plant andalgal materials used to derive fuels like ethanol, butanol, biodiesel,and other hydrocarbon fuels.

As used herein, the phrase “lignocellulosic feedstocks” refers tofeedstocks containing lignocellulose. Non-limiting examples oflignocellulosic feedstocks include, agricultural crop residues (e.g.,wheat straw, barley straw, rice straw, small grain straw, corn stover,corn fibers (e.g., corn fiber gum (CFG), distillers dried grains (DDG),corn gluten meal (CGM)), purpose grown grass crops, energy crops,switchgrass, hay-alfalfa, sugarcane bagasse), corn steep liquor, beetpulp non-agricultural biomass (e.g., algal mats, urban tree residue),corn steep liquor, beet pulp, forest products and industry residues(e.g., softwood first/secondary mill residue, hard softwoodfirst/secondary mill residue, recycled paper pulp sludge),lignocellulosic containing waste (e.g., newsprint, waste paper, brewinggrains, municipal organic waste, yard waste, clinical organic waste,waste generated during the production of biofuels (e.g., processed algalbiomass, glycerol, residues from the production of cellulosic ethanol,solid residues from biodiesel production), and a combination thereof.

As used herein, unless otherwise specified, the term “carbohydrate”refers to a compound of carbon, hydrogen, and oxygen that contains aaldehyde or ketone group in combination with at least two hydroxylgroups. The carbohydrates of the present invention can also beoptionally substituted or deoxygenated at one or more positions.Carbohydrates thus include substituted and unsubstitutedmonosaccharides, disaccharides, oligosaccharides, and polysaccharides.The saccharide can be an aldose or ketose, and may comprise 3, 4, 5, 6,or 7 carbons. In one embodiment they are monosaccharides. In anotherembodiment they can be pyranose and furanose sugars. They can beoptionally deoxygenated at any corresponding C-position, and/orsubstituted with one or more moieties such as hydrogen, halo, haloalkyl,carboxyl, acyl, acyloxy, amino, amido, carboxyl derivatives, alkylamino,dialkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfonic acid,thiol, imine, sulfonyl, sulfanyl, sufinyl, sulfamonyl, ester, carboxylicacid, amide, phosphonyl, phosphinyl, phosphoryl, thioester, thioether,oxime, hydrazine, carbamate. These saccharide units can be arranged inany order and the linkage between two saccharide units can occur in anyof approximately ten different ways. As a result, the number ofdifferent possible stereoisomeric oligosaccharide chain is enormous. Inone embodiment, said carbohydrates are selected from the groupconsisting of monosaccharides, disaccharides, oligosaccharides,polysaccharides, and a combination thereof.

As used herein, the term “monosaccharide” refers to sugar monomersselected from the group consisting of three-carbon sugars (trioses),four-carbon sugars (tetroses), five-carbon sugars (pentoses), six-carbonsugars (hexoses), etc., and a combination thereof. In one embodiment,the five-carbon sugars are selected from the group consisting ofketopentose (e.g., ribulose, xylulose), aldopentose (ribose, arabinose,xylose, lyxose), deoxy sugar (deoxyribose), and a combination thereof.In one embodiment, the six-carbon sugars are selected from the groupconsisting of aldohexoses (e.g., allose, altrose, glucose, mannose,idose, galactose, talose), cyclic hemiacetals, ketohexoses (e.g.,psicose, fructose, sorbose, tagatose). In one embodiment, saidmonosaccharides are selected from the group consisting of trioses,tetroses, pentoses, hexoses, heptoses, etc., and a combination thereof.

In one embodiment, the monosaccharides are in linear form; in anotherembodiment, the monosaccharides are in cyclic form.

As used herein, the phrase “fermentable sugars” refers to sugarcompounds that can be converted to useful value-added fermentationproducts, non-limiting examples of which include amino acids, proteins,sugars, carbohydrates, lipids, nucleic acids, polyketides, vitamins,pharmaceuticals, animal feed supplements, specialty chemicals, chemicalfeedstocks, plastics, solvents, fuels, or other organic polymers, lacticacid, and ethanol. Specific value-added products that may be produced bythe methods disclosed, but are not limited to, β-glucan, lactic acid;specialty chemicals; organic acids, including citric acid, succinic acidand maleic acid; solvents; fish feed and animal feed supplements;pharmaceuticals; vitamins; amino acids, such as lysine, methionine,tryptophan, threonine, carotenoids, human food, nutraceutical, andaspartic acid; industrial enzymes, such as proteases, cellulases,amylases, glucanases, lactases, lipases, lyases, oxidoreductases,transferases and xylanases; and chemical feedstocks.

As used herein, the term “fungus” or “fungi” refers to a distinct groupof eukarotic, organisms with absorptive nutrition and lackingchlorophyll.

As used herein, the term “acidification material” refers to anymaterials, chemical compounds, agents, and/or compositions which whenadded into a solvent (e.g., water), gives a solution with a hydrogen ionactivity greater than in pure solvent (e.g., water). The material can bein gas, liquid, or solid form. The material can be organic and/orinorganic. Non-limiting examples of acidification material include anymaterial that comprises hydrogen halides and their solutions (e.g.,hydrochloric acid (HCl), hydrobromic acid (HBr), and hydroiodic acid(HI)), halogen oxoacids (e.g., hypochloric acid, chloric acid,perchloric acid, periodic acid and corresponding compounds for bromineand iodine), sulfuric acid (H₂SO₄), fluorosulfuric acid, nitric acid(HNO₃), phosphoric acid (H₃PO₄), fluoroantimonic acid, fluoroboric acid,hexafluorophosphoric acid, chromic acid (H₂CrO₄), sufonic acids,methanesulfonic acid (aka mesylic acid, MeSO₃H), ethaneslfonic acid (akaesylic acid, EtSO₃H), benzenesulfonic acid (aka besylic acid, C₆H₅SO₃H),p-toluenesulfonic acid (aka tosylic acid CH₃C₆H₄SO₃H),trifluoromethanesulfonic acid (aka triflic acid, CF₃SO₃H), carboxylicacids (e.g., acetic acid, citric acid, formic acid, gluconic acid,lactic acid, oxalic acid, tartaric acid, Vinylogous carboxylic acids(e.g., ascorbic acid, meldrum's acid), acid salts (e.g., sodiumbicarbonate (NaHCO₃), sodium hydrosulfide (NaHS), sodium bisulfate(NaHSO₄), monosodium phosphate (NaH₂PO₄), and disodium phosphate(Na₂HPO₄)).

As used herein, the term “neutralize,” “neutralizing,” and“neutralization” refers to a chemical reaction in aqueous solutions,wherein an acid and a base react to form water and salt, and wherein thepH of the solution is brought back to an initial pH.

As used herein, the term “manganese donor” refers to a composition orcompound which can provide manganese ion (e.g., manganese (I), manganese(II), and manganese (III)) in an aqueous solution. Non-limiting examplesof manganese donors include, Mn₂(CO)₁₀, K₅Mn(CN)₆NO, MnCl, MnF₂, MnBr₂,MnO, MnO₂, MnCh, MnF₃, MnBr₃, MnCO₃, Mn(CH₃COO)₂, C₆H₉MnO₆, MnTiO₃,[CH₃COCH═C(O)CH₃]₂Mn, [C₆H₁₁(CH₂)₃CO₂]₂Mn, (HCO₂)₂Mn, Mn(C₅HF₆O₂)₂,Mn(PH₂O₂)₂, MnI, (C₃H₅O₃)₂Mn, MnMoO₄, Mn(NO₃)₂, Mn(ClO₄)₂, C₃₂H₁₆MnN₈,MnSO₄, (CH₃COO)₃Mn, C₃₂H₁₆ClMN₈, C₄₈H28ClMN4O₈, C₅H₄CH₃Mn(CO₃),Mn(C₅H₄C₂H₅)₂, and C₁₆H₂₂Mn.

As used herein, the term “pH buffering materials” refers to thecompositions that when added in a liquid mixture, can maintain the pH ofsaid liquid mixture wherein the pH is kept around about 0.5, about 0.6,about 0.7, about 0.8, about 0.9, about 1.0, about 1.1, about 1.2, about1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9,about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about2.6, about 2.7, about 2.8, about 2.9, about 3.0, about 3.1, about 3.2,about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about3.9, about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5,about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8,about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0. For example,the pH of the liquid mixture is in a range between about 0.5 to about3.0. The preferred pH for the filamentous acidophic MK7 strain is about2.2 to about 3.0. Such composition can comprise compounds such as acid,acid salts, basic and basic salts, for example, HCl, H₂NO₃, H₂SO₄,NaHCO₃, NaHS, NaHSO₄, NaH₂PO₄, Na₂HPO₄, NaHSO₃, KHCO₃, KHS, KHSO₄,KH₂PO₄, K₂HPO₄, KHSO₃. NaOH, KOH, Mg(OH)₂, Na₂CO₃, K₂CO₃, KHCO₃, CaCO₃,MgCO₃, Na₂S, K₂S, etc.

As used herein, the term “aerobic conditions” refers to conditions wheresufficient oxygen, is provided, and anaerobic respiration in amicroorganism growing under such conditions is prohibited and anaerobicmetabolic pathways are inhibited preventing anaerobic respiration.

As used herein, the term “microaerobic” and “microaerophilic” are usedinterchangeably to refer to conditions wherein the supply of oxygen islimited, but the cellular respiration in an organism is dominantlyaerobic respiration.

As used herein, the term “fatty acids” refers to long-chained moleculeshaving a methyl group at one end and a carboxylic acid group at theother end.

As used herein, the term “isolated fungus” refers to any compositioncomprising a fungus population which is obtained from a natural source.

As used herein, the term “carbon source” generally refers to a substancesuitable to be used as a source of carbon for prokaryotic or eukaryoticcell growth. Carbon sources include, but are not limited to, biomasshydrolysates, acid whey, sweet whey, carbohydrates (e.g., starch,sucrose, polysaccharides, and monosaccharides), cellulose,hemicellulose, xylose, and lignin, as well as monomeric components ofthese substrates and/or combinations thereof. Carbon sources cancomprise various organic compounds in various forms, including but notlimited to polymers, carbohydrates, acids, alcohols, aldehydes, ketones,amino acids, peptides, etc. These include, for example, variousmonosaccharides such as glucose, dextrose (D-glucose), maltose,oligosaccharides, polysaccharides, saturated or unsaturated fatty acids,succinate, lactate, acetate, ethanol, etc., or mixtures thereof. Acarbon source can also be a feedstock or lignocellulosic feedstock, suchas sugar beet pulp. Photosynthetic organisms can additionally produce acarbon source as a product of photosynthesis.

As used herein, the term “biocatalyst” refers to a living system or cellof any type that speeds up chemical reactions by lowering the activationenergy of the reaction and is neither consumed nor altered in theprocess. Biocatalysts may include, but are not limited to,microorganisms such as yeasts, fungi, bacteria, and archaea. Forexample, the isolated fungal species and/or strain(s) of the presentinvention can be used as a biocatalyst in the production of proteins andlipids, or in the degradation of carbon substrates or organic moleculesfor the production of proteins and lipids.

As used herein, the term “fermentation” or “fermentation process” refersto a process in which an organism or a biocatalyst is cultivated in aculture medium containing raw materials, such as a carbon source andnutrients, wherein the organism or biocatalyst converts those rawmaterials into products.

As used herein, the term “biomass” refers to biological material derivedfrom living, or recently living organisms, e.g., stems, leaves, andstarch-containing portions of green plants, or wood, waste, forestresidues (dead trees, branches and tree stumps), yard clippings, woodchips, or materials derived from algae or animals and/or industrialbyproducts and waste streams, food waste/scraps, and other simplesugars. In some cases, biomass contains a significant portion of proteinand/or lipid. In other cases, it is mainly comprised of starch, lignin,pectin, cellulose, hemicellulose, and/or pectin.

As used herein, the term “cellulosic biomass” refers to biomass composedprimarily of plant fibers that are inedible or nearly inedible by humansand have cellulose as a prominent component. Those fibers may behydrolyzed to yield a variety of sugars that can be fermented bymicroorganisms. Examples of cellulosic biomass include grass, wood, andcellulose-rich residues resulting from agriculture or the forestproducts industry.

As used herein, the terms “filamentous biomat,” and “filamentous fungibiomat” are used interchangeably and refer to biomats produced by andcontaining filamentous fungi.

As used herein, the term “starch” refers to a polymer of glucose readilyhydrolyzed by digestive enzymes, e.g., amylases. Starch is usuallyconcentrated in specialized portions of plants, such as potatoes, cornkernels, rice grains, wheat grains, and sugar cane stems.

As used herein, the term “lignin” refers to a polymer material, mainlycomposed of linked phenolic monomeric compounds, such as p-coumarylalcohol, coniferyl alcohol, and sinapyl alcohol, which forms the basisof structural rigidity in plants and is frequently referred to as thewoody portion of plants. Lignin is also considered to be thenon-carbohydrate portion of the cell wall of plants.

As used herein, the term “cellulose” refers to a long-chain polymerpolysaccharide carbohydrate of beta-glucose of formula (C₆H₁₀O₅)_(n),usually found in plant cell walls in combination with lignin and anyhemicellulose.

As used herein, the term “hemicellulose” refers to a class of plantcell-wall polysaccharides that can be any of several heteropolymers.These include xylan, xyloglucan, arabinoxylan, arabinogalactan,glucuronoxylan, lucomannan and galactomannan. Monomeric components ofhemicellulose include, but are not limited to: D-galactose, L-galactose,D-rnannose, L-rhamnose, L-fucose, D-xylose, L-arabinose, andD-glucuronic acid. This class of polysaccharides is found in almost allcell walls along with cellulose. Hemicellulose is lower in weight thancellulose and cannot be extracted by hot water or chelating agents, butcan be extracted by aqueous alkali. Polymeric chains of hemicellulosebind pectin and cellulose in a network of cross-linked fibers formingthe cell walls of most plant cells.

The term “pectin” as used herein refers to a class of plant cell-wallheterogeneous polysaccharides that can be extracted by treatment withacids and chelating agents. Typically, 70-80% of pectin is found as alinear chain of α-(1-4)-linked D-galacturonic acid monomers. The smallerRG-I fraction of pectin is comprised of alternating (1-4)-linkedgalacturonic acid and (1-2)-linked L-rhamnose, with substantialarabinogalactan branching emanating from the rhamnose residue. Othermonosaccharides, such as D-fucose, D-xylose, apiose, aceric acid, Kdo,Dha, 2-O-methyl-D-fucose, and 2-O-methyl-D-xylose, are found either inthe RG-II pectin fraction (<2%), or as minor constituents in the RG-Ifraction. Proportions of each of the monosaccharides in relation toD-galacturonic acid vary depending on the individual plant and itsmicro-environment, the species, and time during the growth cycle. Forthe same reasons, the homogalacturonan and RG-I fractions can differwidely in their content of methyl esters on GalA residues, and thecontent of acetyl residue esters on the C-2 and C-3 positions of GalAand neutral sugars.

As used herein, the term “facultative anaerobic organism” or facultativeanaerobic microorganism” or a “facultative anaerobic biocatalyst” isdefined as an organism that can grow in wither the presence or in theabsence of oxygen, such as the fungal strains isolated in the presentinvention.

As used herein, the term “distillers dried grains”, abbreviated as DDG,refers to the solids remaining after a fermentation, usually consistingof unconsumed feedstock solids, remaining nutrients, protein, fiber, andoil, as well as biocatalyst cell debris. The term may also includesoluble residual material from the fermentation and is then referred toas “distillers dried grains and solubles” (DDGS).

As used herein, the term “nutrient” is defined as a chemical compoundthat is used by an organism or biocatalyst to grow and survive. As anexample, nutrients can be organic compounds such as carbohydrates andamino acids or inorganic compounds such as metal salts.

As used herein, the term “complex nutrient” is defined a nutrient sourcecontaining mostly monomeric organic compounds used by an organism orbiocatalyst for the production of proteins, DNA, lipids, andcarbohydrates. The term “rich nutrient” is used interchangeablythroughout with the term complex nutrient. Typically, complex nutrientsor rich nutrients are derived from biological materials, such asslaughterhouse waste(s), dairy waste(s), or agricultural residues.Complex nutrients or rich nutrients include, but are not limited to:yeast extract, tryptone, peptone, soy extract, corn steep liquor, soyprotein, and casein.

As used herein, the term “aerobic metabolism” refers to a biochemicalprocess in which oxygen is used to make energy, typically in the form ofATP, from carbohydrates. Typical aerobic metabolism occurs viaglycolysis and the TCA cycle, wherein a single glucose molecule ismetabolized completely into carbon dioxide in the presence of oxygen.

As used herein, the phrase “anaerobic metabolism” refers to abiochemical process in which oxygen is not the final acceptor ofelectrons contained in NADH. Anaerobic metabolism can be divided intoanaerobic respiration, in which compounds other than oxygen serve as theterminal electron acceptor, and fermentation, in which the electronsfrom NADH are utilized to generate a reduced product via a “fermentativepathway.”

As used herein, the term “microbiological fermentation” refers to aprocess where organic substances are broken down and re-assembled intoproducts by microorganisms. The substances may include, but are notlimited to, glucose, sucrose, glycerol, starch, maltodextrine, lactose,fats, hydrocarbons, protein, ammonia, nitrate, and phosphorus sources.The products may include, but not limited to, specialty products(including but not limited to, mycoprotein products, soy products,tempeh, etc.), traditional products (including but not limited to,bread, beer, wine, spirits, cheese, dairy products, fermented meats andvegetables, mushrooms, soy sauce and vinegar), agricultural products(including but not limited to, gibberellins, fungicides, insecticides,silage, amino acids such as L-Glutamine, L-Lysine, L-Tryptophan,L-Throenine, L-aspartic (+), L-arylglycines), enzymes (including but notlimited to carbohydrates, celluloses, lipases, pectinases, proteases),fuels and chemical feedstocks (including but not limited to, acetone,butanol, butanediol, isopropanol, ethyl alcohol, glycerol, methane,glycerol, butyric acid, methane, citric acid, fumaric acid, lactic acid,propionic acid, succinic acid, and L-glutaric acid or salts of any ofthese acids), nucleotides, organic acids, pharmaceuticals and relatedcompounds (including but not limited to alkaloids, antibiotics,hormones, immunosuppressant, interferon, steroids, vaccines, vitamins)and polymers (including but not limited to alginates, dextran, gellan,polyhydroxybutyrate, scleroglucan and xanthan). The microorganisms usedfor fermentation may include both prokaryotic microorganisms (includingbacteria, cyanobacteria) and eukaryotic microorganisms (including yeast,fungi and algae).

As used herein, the phrase “energy crops” refers to plants grown as alow cost and low maintenance harvest used to make biofuels, or directlyexploited for its energy content. Commercial energy crops are typicallydensely planted, high yielding crop species where the energy crops willbe burnt to generate power. Woody crops such as Willow or Poplar arewidely utilized as well as tropical grasses such as Miscanthus andPennisetum purpureum (bout known as elephant grass).

As used herein, the term “surface fermentation” refers to thosefermentations in which the microorganisms employed grow on the surfaceof the fermentation media without any further support. The media istypically a free-flowing aqueous media. Without being bound by theory,it is thought that filamentous biomats result from some combination ofaerobic, microaerobic and/or anaerobic metabolism. For example, thesurface of the biomat is thought to rely on aerobic respiration whilethe bottom of the biomat may be microaerobic to highly anaerobic.

As used herein, the term “solid substrate surface fermentation” refersto those fermentations in which the microorganisms employed grow on thesurface of the fermentation media using carbon and nutrients supplied bysolids that are submerged in the fermentation media. In someembodiments, some portion of the biomat may be partially submerged.

As used herein, the term “submerged fermentation” refers to thosefermentations wherein the microorganisms employed grow in a submergedstate within fermentation media. Many fermentations fall within thiscategory, such as the penicillin submerged fermentation technique.

As used herein, the term “solid-state fermentation” refers to theculture of microorganisms grown on a solid support selected for thepurpose. For example, a solid culture substrate, such as rice or wheatbran, is deposited on flatbeds after seeding with microorganisms; thesubstrate is then left in a temperature-controlled room for severaldays. Solid-state fermentation uses culture substrates with low waterlevels (reduced water activity). The medium (e.g. rice or wheat bran) issaturated with water, but little of it is free flowing. The solid mediumcomprises both the substrate and the solid support on which thefermentation takes place.

As used herein, the term “nutraceutical” refers to substances that havehealth or medicinal benefits. In some instances, a nutraceutical notonly supplements the diet but also aids in the prevention and/ortreatment of disease and/or disorders. The term “nutraceutical” wascoined from “nutrition” and “pharmaceutical” in 1989 by Stepen DeFelice,Md., founder and chairman of the Foundation for Innovation in Medicine(FIM).

As used herein, “progeny” refers to any and all descendants by lineagewhich originate from a strain no matter however or wherever produced.Included within the definition of “progeny” as used herein are any andall mutants of the isolated/deposited strain and its progeny, whereinsuch mutants have at least one of the physiological and/or morphologicalcharacteristics of the isolated/deposited strain and its progeny.

Artificial Media for Growth of Filamentous Fungi Biomat

An artificial media is used to produce a filamentous fungal biomat. Theartificial media provides the nutrients required for increased cellcycle times as compared to those found in nature (i.e. increased growthrate) and results in increased cell density. The artificial mediacomprises at least the following macronutrients: nitrogen (N),phosphorus (P), calcium (Ca), magnesium (Mg), carbon (C), potassium (K),sulfur (S). Trace nutrients such as iron (Fe), boron (B), chromium (Cr),copper (Cu), selenium (Se), manganese (Mn), molybdenum (Mo), vanadium(V), and zinc (Zn) can also be added to the media to supplement carbonsources. Carbon sources such as lignocellulosic feedstocks, sweet whey,and/or acid whey typically provide sufficient trace nutrients so thatadditional trace nutrients are not required.

Additional nutrient additions can be added to the artificial media.Examples of such are carbohydrates (e.g., monosaccharides,polysaccharides), amino acid donors (e.g., amino acid, polypeptides),and combinations thereof. In addition, compounds that can facilitatepretreatment of the lignocellulosic carbon source can also be added intothe artificial media. Such compounds include, but are not limited to,acidification materials, manganese donors, nutrients, and pH bufferingmaterial.

The artificial medium can be in the form of a liquid impregnated solid,a liquid, or a gel. The artificial medium can also be in the form of aliquid covering a solid carbon substrate, such as a lignocellulosefeedstock or other solid carbon substrate. Here, the solid substrate issubmerged under the surface of a liquid, such that the biomat grows onthe surface of the liquid using carbon derived from the submerged solid,a process known as solid substrate surface fermentation (SSSF).Extracellular enzymes excreted from the fungus degrade the solid carbonsubstrate, releasing soluble carbon that can be taken up by the biomatat or near the biomat/water interface. The resulting biomat forms a maton a liquid layer above the submerged solid substrate. In general, theliquid layer above the submerged carbon source should be about 0.01-1.0cm deep. Too little liquid results in no mat formation and solid-statefermentation and/or submerged fermentation ensues. Too much liquidresults in inefficient conversion and a depressed biomat growth cycle.

There are a large variety of substances that can be used as a carbonsource for the artificial media. These include sugars (e.g., glucose,galactose, mannose, trehalose, sucrose, arabinose, mannose, xylose,fructose, etc.), glycerol, starch, carbohydrates, glycerol, whey,lignocellulosic feedstock, waste stream(s) (e.g. acid whey) andcombinations thereof. Suitable lignocellulosic feedstocks include, forexample, switchgrass, energy crops, forest hardwoods and other products,brewers spent grain, wheat straw, grasses, leaves, AFEX crop residues,anaerobic digestate, agricultural crop residues (e.g., barley straw,rice straw, small grain straw, corn stover, corn fibers (e.g., cornfiber gum (CFG), distillers dried grains (DDG), corn gluten meal (CGM)),hay-alfalfa, sugarcane bagasse, non-agricultural biomass (e.g., algalmats, urban tree residue), industry residues (e.g., softwoodfirst/secondary mill residue, hard softwood, first/secondary millresidue, recycled paper, pulp sludge), lignocellulosic containing waste(e.g., newsprint, waste paper, brewing grains, municipal organic waste,yard waste), clinical organic waste, waste generated during theproduction of biofuels (e.g., processed algal biomass, residues from theproduction of cellulosic ethanol, solid residues from biodieselproduction), and a combination thereof. Suitable waste stream(s) includeagricultural waste, municipal organic wastes, waste from biofuelproduction (e.g., cellulosic ethanol production residues), algalbiomass, brewers spent grain and/or waste streams (e.g., molasses, cornsyrup, etc.), industrial waste (e.g., organic molecules such as phenoland other aromatics) and fibers such as beta-glucan, cellulose, chitin,hemicellulose and polydextros, monosaccharides, disaccharides,oligosaccharides, polysaccharides, and any combination thereof. Themonosaccharides encomplass trioses, tetroses, pentoses, hexoses,heptoses, etc., and any and all combinations thereof, the pentosesencompass ribulose, xylulose, ribose, arabinose, xylose, lyxose,deoxyribose, and any and all combinations thereof, while the hexoses areselected from the group consisting of allose, altrose, glucose, mannose,glucose, idose, galactose, talose, psicose, fructose, sorbose, tagatose,and any and all combinations thereof. The disaccharides encompasssucrose, lactose, maltose, and any and all combinations thereof whilethe polysaccharides encompass starch, glycogen, cellulose, chitin, andany and all combinations thereof.

The carbon source that is used to grow the isolated fungal strain cancomprise cellulose in an amount of from about 5% to about 100%, fromabout 10% to about 95%, from about 20% to about 90%, from about 30% toabout 85%, from about 40% to about 80%, from about 50% to about 75%, orfrom about 60% to about 70% by dry weight of the carbon source.Alternatively, the cellulosic carbon source comprises cellulose in anamount of at least about 5%, at least about 10%, at least about 20%, atleast about 30%, at least about 40%, at least about 50%, at least about60%, or at least about 70% of the dry weight of the carbon source. Inother cases, the cellulosic carbon source used to grow the isolatedfungal strain comprises from about 1% to about 50%, from about 5% toabout 40%, or from about 10% to about 30% by weight of a componentselected from lignin, hemicellulose, or a combination thereof. In someembodiments of the present invention, the cellulosic carbon source usedto grow a microorganism comprises at least about 1%, at least about 5%,at least about 10%, at least about 20%, or at least about 30% by weightof a component selected from lignin, hemicellulose, or a combinationthereof.

Suitable nitrogen sources include urea, ammonium nitrate (NH₄NO₃),ammonium sulfate (NH₄SO₄), nitrate salts (e.g. KNO₃), ammonia salts(i.e. NH₄SO₄) and organic N (e.g. proteins, peptides), industrial wastestreams high in nitrogen, corn steep liquor, and combinations thereof.Artificial media prepared with a pure urea nitrogen source providesapproximately 25% faster growth of the filamentous fungi than doesartificial media prepared with a combination of urea and ammoniumnitrate (i.e. 70 g/m²/day vs. 52 g/m²/day, respectively). Combinationsof urea and ammonium nitrate can also be used. Growth, albeit muchslower than that produced with urea alone or a urea combination, alsooccurs when ammonium sulfate is used as the sole nitrogen source. Whileammonium nitrate alone can also be used, this nitrogen source again doesnot produce the vigorous growth seen with combinations of urea.

Manipulation of the carbon to nitrogen ration (C:N) within theartificial media has a significant influence on the composition of thebiomat produced by the fungal species and/or strain(s). Typically, a lowC:N ratio, such as a C:N ratio of 7.5:1 or less, favors production ofproteins and amino acids as compared to lipids. On the other hand, a C:Nratio of more than 7.5:1 favors production of lipids as compared toproteins. Oftentimes lipid formation is particularly favored when theartificial media has a C:N ratio of at least 10:1, 15:1, 20:1, 26:1,30:1, 40:1, or 50:1.

The pH of the artificial media is determined based on the productsdesired and the fungal species and/or strain(s) employed. Fusisporium,Pseudofusarium, Gibberella, Sporotrichella, Aspergillus, Penicillium,Triocoderma, species within the Mucorales sp. (e.g., Rhizopus sp.), theisolated filamentous acidophilic fungal strain designated as MK7, andcombinations thereof, high lipid production takes place over pH range2.0-7.0 and optimally at a pH of less than 3.5. High protein production,while predominantly influenced as a function of C:N ratios, requires apH of at least 2.7 and preferably a pH between 4.5 and 5.5.

Cultures and Compositions Comprising Isolated Fungal Species and/orStrains

The present invention uses a pure culture of an isolated fungal speciesand/or strain, or a pure co-culture of two fungal species and/orstrains, or comprised of a substantially pure culture of three or morefungal species and/or strains. A large number of isolated filamentousfungal species and/or strains can be used, such as a species and/orstrain(s) of Fusisporium, Psedofusarium, Gibberella, Sporotrichella,Aspergillus. Penicillium, Triocoderma, Pichia spp, species within theMucorales sp. (e.g. Rhizopus sp.), and combinations thereof. Thebiologically pure culture/co-culture/substantially pure culture can alsocomprise the isolated filamentous acidophilic fungal strain designatedas MK7, which has been deposited as ATCC Accession Deposit No.PTA-10698, or active mutants thereof. Biologically pure cultures ofgenetically modified filamentous fungi can also be used. The pure fungalspecies and/or strain(s) and/or its progeny are typically in the form ofconidia, microconidia, macroconidia, pycnidia, chlamydospores, hyphae,fragments of hyphae and mycelia or a combination thereof.

The filamentous acidophilic MK7 fungal strain is a new strain ofacidophilic fungus, which can directly convert carbon sources such aslignocellulosic carbon sources, carbohydrates, (e.g., acid whey) andalgal biomass to filamentous fungi biomats comprising proteins andlipids.

Methods of producing useful products using the artificial media and theisolated fungus stain and/or its progeny, comprise:

-   -   a) Inoculating one or more of the fungal species or strains        and/or its progeny into artificial media having a carbon source        selected from the group consisting of sugar, glycerol,        lignocellulosic feedstocks, carbon containing agricultural,        industrial, and municipal waste products, carbohydrates, yeast        extract, casamino acids, acid whey, sweet whey and/or a        combination thereof in a container, wherein the artificial media        can support the growth of said isolated fungal strain via        surface fermentation;    -   b) growing said isolated fungal strain in said artificial media        to produce filamentous fungi biomats;    -   c) harvesting the filamentous fungi biomats; and    -   d) optionally isolating, purifying and/or producing products        from the filamentous fungi biomats.

Growth is produced under aerobic conditions. In another embodiment thegrowth is produced under microaerobic conditions. Alternatively, growthis produced as the result of any combination of aerobic conditions,microaerobic conditions and anaerobic conditions, such as via surfacefermentation.

The useful products are protein-rich biomass, biomats and/or afilamentous fungi biomat. For example, the useful products producedusing the fungi and methods disclosed include, but are not limited to,protein biomats for use in food products, fish feed products, animalfeed products, bioplastics, and/or precursors thereof. Here, the growthis produced by aerobic conditions, microaerobic conditions, andanaerobic conditions or any combination thereof.

A large number of the acidophilic fungal species and/or strain(s), suchas Fusisporium, Pseudofusarium, Gibberella, Sporotrichella, Aspergillus.Penicillium, Triocoderma, species within the Mucorales sp. (e.g.,Rhizopus sp.), the isolated filamentous acidophilic fungal straindesignated as MK7 and combinations thereof, and/or their progeny can becultured in the absence of antibiotics with little or no contamination.Typically, contamination in the artificial media is caused by otherorganisms such as bacteria, other undesired fungi (e.g., yeasts, molds),algae, plants, insects, and a mixture thereof.

At least one composition comprising an isolated fungal species and/orstrain of Fusisporium, Pseudofusarium, Gibberella, Sporotrichella,Aspergillus. Penicillium, Triocoderma, species within the Mucorales sp.(e.g., Rhizopus sp.), yeasts capable of producing filaments (i.e.Yarrowia) the isolated filamentous acidophilic fungal strain designatedas MK7, and combinations thereof is also disclosed. The composition canfurther comprise an artificial medium that supports growth of the fungalspecies and/or strain(s), and optionally one or more of an acidificationmaterial, a manganese donor, a nutrient addition, and/or a mixturethereof.

Surface Fermentation

The current disclosure initiates surface fermentation by inoculatingartificial media with a suspension of planktonic cells of the desiredfilamentous fungal species and/or strain(s). Inoculum culture from aninoculum reactor is added to the artificial media at a concentrationthat will produce a mature biomat in the desired period of time. Intheory, the media could be inoculated with a single cell; however, suchan inoculation would require extraordinarily stringent sterilityconditions and a significantly extended period of time in order formature biomat to develop. Typically, inoculation with 0.5-1.0 g of cellsper liter of growth media will produce a biomat in 3 to 6 days. Forexample, adding inoculum containing about 10 g/L of cells at 7.5%(volume to volume) of the medium used will produce a biomat in 3 to 6days. No external oxygen is introduced to the artificial media bybubbling or other means, sufficient oxygen can be culled from ambient ornear ambient conditions.

Without being bound by theory, it is thought that because cell growth ismuch more rapid in the presence of oxygen, conidia present at thesurface of the artificial media where more oxygen is present will growrapidly and begin formation of the mycelial biomat. It is believed thatoxygen concentrations are much lower only a few micrometers below thesurface of the artificial media and consequently would place fungalcells located in those regions in a stress environment. Stress is knownto increase excretion of extracellular polysaccharides, which have a“sticky” phenotype, and would thus aid in the rapid formation of thefilamentous fungi biomat by adhering to the cells proliferating at thesurface. Substrate concentration, however, also has a significanteffect. For example, when the carbon substrate concentration is below4%, filamentous fungi biomats will not form. It should be noted thatinitial environmental stress to form mats does not necessarily inferthat a stressed mat, i.e. a mat containing toxins excreted by thestressed organism is formed.

Typically, shallow trays containing artificial media are used forsurface fermentation under controlled conditions of temperature,humidity, and airflow suitable for the fungal species and/or strains(s)employed. Sterile conditions are maintained for optimal filamentousfungi biomat growth. Sufficient airflow is maintained to remove heat andcarbon dioxide produced from microbial respiration and supply oxygenwithout agitating the surface of the artificial media and disruptingfungal hyphae growth.

In general, a “skin” begins to form on the surface of the artificialmedia on day 2 after inoculation. This “skin” is the initial filamentousfungi biomat which frequently includes aerial hyphae as well as hyphaein contact with the artificial media and which continues to grow andincrease in cell density. Typically, three to six days afterinoculation, the resultant filamentous fungi biomats are 1 to 30 mmthick and have sufficient tensile strength and structural integrity tobe handled without tearing.

The filamentous fungi biomats produced have a structure as describedwhich is not seen in nature. First, naturally formed filamentous fungibiomats are not composed of a pure culture/co-culture/substantially pureculture. Typically, the biomats formed in nature contain various typesof algae and/or bacteria in addition to at least one filamentous fungalspecies and form an artificial microecosystem. Examples of fungalbiomats formed in nature are mycorrhizal fungal mats, which exist in alargely dispersed form in the soil and are associated with plant roots,lichens (e.g. Reindeer moss and crustose lichen), and mushrooms (e.g.Armillaria ostoyae).

Second, the biomats formed using the methods and techniques describedherein have a significantly greater cell density than those found innature, even taking into account the multiple species found in naturallyformed biomats. The produced filamentous fungi biomats tend to be verydense typically, 50-200 grams per liter. Natural and submerged processesfor growth of filamentous fungi commonly result in biomass densities ofabout, 15 grams per liter. Solid-state fermentation processes result ina mixture of the substrate with a small percentage of fungi, i.e. lessthan 5% fungal composition. From the perspective of percent solids, themethods disclosed herein produced filamentous fungi biomats thatcommonly range from 5-20% solids. In contrast, natural and submergedprocesses for growth of filamentous fungi commonly result in percentsolid ranges of less than 1.5%. One result of the densities achieved,the filamentous nature, and the extracellular matrix found in thesedense biomats is an ability to be maintained as a cohesive mat upondrying. This is in stark contrast to the powdery and/or non-cohesiveform normally found with other dried filamentous fungi biomats.

Third, the biomats formed using the methods and techniques describedherein have a high tensile strength compared to naturally occurringbiomats, allowing them to be lifted and moved without breakage.

Fourth, the instant biomats have a defined structure comprising, in someinstances, a single dense layer comprised of long filaments generallyaligned parallel to the air:biomat interface. In some filamentous fungibiomats at least two layers exist: (a) a dense bottom layer and (b) anaerial hyphae layer. In some filamentous fungi biomats at least threestructurally different layers are visible: (a) a dense bottom layer, (b)an aerial hyphae layer and (c) a transition zone layer (see FIGS. 10Aand B). For systems with aerial hyphae and systems with three layers,the aerial hyphae layer is typically most visibly dominant, followed bythe dense bottom layer, while the transition zone layer, if present, issmallest. Each of the layers normally has a characteristic cell densityassociated with it as compared to the other layer(s). For example, theaerial hyphae layer is significantly less dense than the bottom layer ofthe biomat (see FIG. 10A). If aerial hyphae are produced, they arepredominantly oriented perpendicular to the biomat:air and/orbiomat:media interface. For all biomats, the dense layer is comprised oflong filaments which are predisposed to be aligned parallel with thebiomat:air and/or biomat:media interface. Further, the resulting biomatis comprised of at least a majority of the fungal biomass and inpreferred embodiments, comprised of essentially no residual feedstockand is essentially pure fungal biomass.

In those instances where aerial hyphae are formed, such as when glycerolis used as a substrate, a number of key distinguishing factors alsoexist between the aerial hyphae layer and the dense bottom layer. Interms of length, aerial hyphae tend to be longer than those found in thedense bottom layer. The density and distribution of the individualaerial hyphae is less than those associated with the dense layermycelium. The aerial hyphae tend to a vertical orientation at theterminus juxtaposed to the atmosphere. That is, aerial hyphae tend togrow relatively perpendicular to the surface medium. On the other hand,the hyphae of the dense layer tend to grow in a predominantly parallelorientation to the air:biomat and/or biomat:media interface. The lowrelative density of the aerial hyphae combined with their longer lengthand vertical orientation suggests a maximization of oxygen harvesting.Further, little to no extra cellular matrix is found in the aerialhyphae layer. In contrast, a lot of extracellular matrix can be found inthe dense bottom layer.

The aerial layer of the biomat, if formed, appears to accelerate thegrowth of the biomat. Disruptions to the aerial layer disrupted areanegatively impact the accelerated growth of the biomat. Disruptionsinclude contact with a solid object, contact with water droplets, andcracks or fissures caused from agitation of the liquid media upon whichthe biomat grows. Typically, the disrupted biomat area undergoes nofurther growth when the cause of the disruption is removed. Generally,the biomat growth is produced by aerobic conditions, microaerobicconditions, and anaerobic conditions or any combination thereof.

The biomats are normally harvested between day 3 and day 12 afterinoculation, depending on the species/strain(s) used and the desiredproduct, although later harvest times are also possible. The filamentousfungi biomats can be harvested by a number of different methodologieswhich can include; rinsing, physical processing (size reduction,pressure treatments, dehydration, etc.), inactivation of viabilityprocedures, temperature cycling, extractions and/or separation ofbiomass constituents, and conversion and/or inclusion into differentsystems. In some embodiments the filamentous fungi biomats areharvested, rinsed with water, and are then either dried in a temperaturecontrolled oven to deactivate many of the enzymes and limit biochemicaltransformations within the biomat, or, are frozen.

Filamentous fungi are recognized as very useful as host cells forrecombinant protein production and expression platforms, resulting inuseful products expressed in the biomass, and/or a filamentous fungibiomat. Examples of filamentous fungi which are currently used orproposed for use in such processes include Neurospora crassa, Acremoniumchrysogenum, Tolypocladium geodes, Mucor circinelloides, Trichodermareesei, Aspergillus nidulans, Aspergillus niger and Aspergillus oryzae.Further, microbial species used to produce biomats as disclosed can begenetically modified to express/depress systems by manipulation of geneexpression, including transcription, such that they either overexpressor do not express compounds or chemistries found in their native orunaltered form. The use of and manipulation of fungal systems to overexpress existing chemistries, express systems not naturally present ordepress systems commonly present in the native form is known as the art,i.e. Aspergillus spp., Penicillium spp., Rhizopus spp., Trichodermaspp., and yeasts such as Pichia spp. Useful products produced using thebiomat and methods disclosed include, but are not limited to, biomassand/or biomass biomats used to express pharmaceuticals, nutraceuticals,building block chemicals for industrial application, medicinals, enzymesand/or precursors thereof.

Acidophilic Fungal Species and/or Strain(s)

The acidophilic fungal species and/or strains used in the presentinvention are lignocellulose degrading, filamentous fungal strainsand/or their progeny that have at least the following identifyingcharacteristics:

-   -   a) the isolated strain is acidophilic and can grow at pH ranging        from about 0.68 of about 8.5; and    -   b) produce filamentous, biomats containing proteins and lipids        from artificial media via surface fermentation under aerobic        conditions, microaerobic conditions, anaerobic conditions or any        combination thereof. Here, the artificial media's carbon source        includes carbohydrates, lignocellulosic feedstocks, carbon        containing waste products (e.g. acid whey), or a combination        thereof.

The isolated species and/or strain(s) further typically comprise one ormore of the following additional identifying characteristics:

-   -   c) the ability to produce proteins, lipids amino acids, enzymes,        nucleic acids (nucleotides), carbohydrates, fibers such as beta        glucans, polyketides, alkaloids, pigments, and antibiotics.        Examples of include, but are not limited to esters, glutamic        acid, aspartic acid, amylases, proteases, cellulases, xylanases,        lipases, peroxidases, manganese peroxidases, nucleic        acids/nucleotides: DNA/RNA, purines, pyrimidines, oleic acid,        palmitoleic acid, beta-glucan, chitin, beta-carotene,        glycosides, phenolics, terpenoids from carbon sources as        described on page 18, paragraph [80] and algal feedstocks, and        from waste generated during biofuel production (e.g. processed        algal biomass, glycerol) under a variety of anaerobic, aerobic        microaerobic conditions and/or any combination thereof;    -   d) comprise an 18S rRNA and ITS region DNA sequence that shares        at least 98% identity to SEQ ID NO.:1.

Suitable filamentous acidophilic fungal species and/or strain(s) includeFusisporium, Pseudofusarium, Gibberella, Sporotrichella, Aspergillus,Penicillium, Triocoderma, species within the Mucorales sp. (e.g.,Rhizopus sp.), the isolated filamentous acidophilic fungal straindesignated as MK7, and combinations thereof, and/or their progeny. Thestrain designated as MK7, has been deposited as ATCC Accession DepositNo. PTA-10698.

The acidophilic fungal species and/or strain(s) and/or its progeny cangrow at a low pH of at most about 7.0, about 6.5, about 6.0, about 5.5,about 5.0, about 4.5, about 4.0, about 3.5, about 2.0, about 1.8, about1.6, about 1.4, about 1.2, about 1.0, about 0.9, about 0.8, or about 0.7or about 0.6, or about 0.5. For example, the fungal strain can grow at alow pH ranging from about 0.68 to about 2.0.

The acidophilic species and/or strain(s) employed can produce lipids andproteins in high quantities within the filamentous fungi biomats grownunder the low pH ranges as described above. For example, the isolatedstrain can convert the carbon source to lipids at a higher rate within alow pH as described above than has been previously reported in the art,such as the previously isolated Fusarium strains have been described(see Nairn et al., 1985, Bhatia et al., 2006, and Naqvi et al., 1997).The acidophilic species and/or strain(s) employed can convert a carbonsource to lipids at a rate of at least 0.04 g lipid/g carbon source,0.05 g lipid/g carbon source, 0.06 g lipid/g carbon source 0.07 glipid/g carbon source, 0.08 g lipid/g carbon source, 0.1 g lipid/gcarbon source, 0.12 g lipid/g carbon source, 0.14 g lipid/g carbonsource, 0.16 g lipid/g carbon source, 0.18 g lipid/g carbon source, 0.2g lipid/g carbon source, 0.25 g lipid/g carbon source, 0.3 g lipid/gcarbon source, 0.35 g lipid/g carbon source, or 0.4 g lipid/g carbonsource, after 10 days incubation at pH 2.5.

The culturing conditions of the current invention also producesfilamentous biomass having a more favorable lipid profile when comparedto the biomass previously produced from cultured fungi or microalgae.For example, the acidophilic species and/or strain(s) used produce moresaturated fatty acids (e.g., palmitic (16:0) and stearic acids (18:0))and mono-unsaturated fatty acids (e.g., oleic acid (18:1)), but lesspolyunsaturated fatty acids, which area more vulnerable to oxidation.

In addition, the acidophilic fungal species and/or strain(s) and/or itsprogeny can grow at a high metal concentration, where the metal isselected from the group consisting of Mn, Ag, Zn, Fe, Al, Be, Pb, Cu,Cr, Ni, Cd, Co, Ni, Pd, Pt, U, Th, Mo, Sn, Ti, As, Au, Se, Sb and Hg.

The acidophilic fungal species and/or strains and/or their progeny arecapable of rapid, high density cell growth under the culturingconditions. Here, the microorganisms are capable of achieving a celldensity (measured as dry weight/L of artificial media) of at least about10 g/L, at least about 15 g/L, at least about 20 g/L, at least about 25g/L, at least about 30 g/L, at least about 50 g/L, at least about 75g/L, at least about 100 g/L, at least about 125 g/L, at least about 135g/L, at least about 140 g/L, at least about 145 g/L, at least about 150g/L, at least about 160 g/L, at least about 170 g/L, at least about 180g/L, at least about 190 g/L, at least about 200 g/L, at least about 210g/L, at least about 220 g/L, at least about 230 g/L, at least about 240g/L, at least about 250 g/L.

For example, the acidophilic fungal species and/or strain(s) are capableof achieving a cell density of from about 10 g/L to about 300 g/L, fromabout 15 g/L to about 300 g/L, from about 20 g/L to about 300 g/L, fromabout 25 g/L to about 300 g/L, from about 30 g/L to about 300 g/L, fromabout 50 g/L to about 300 g/L, from about 75 g/L to about 300 g/L, fromabout 100 g/L to about 300 g/L, from about 125 g/L to about 300 g/L,from about 150 g/L to about 300 g/L, from about 170 g/L to about 300g/L, from about 130 g/L to about 290 g/L, from about 135 g/L to about280 g/L, from about 140 g/L to about 270 g/L, from about 145 g/L toabout 260 g/L, from about 150 g/L to about 250 g/L, from about 170 g/Lto about 250 g/L, from about 100 g/L to about 280 g/L. The high densitygrowth of the acidophilic fungal species and/or strain(s) of can befurther increased by adjusting the fermentation conditions (such astemperature, pH, concentration of ions, time of incubation and/or gasconcentrations).

Fusarium Species

Information regarding acidophilic Fusarium species, methods ofidentifying, isolating culturing is described in Nelson et al.,(Taxonomy, Biology, and Clinical Aspects of Fusarium Species, 1994,Clinical Microbiology Reviews, 7(4): 479-504), Toussoun and Nelson(1976, Fusarium), Booth (Fusarium: laboratory guide to theidentification of the major species, 1977, Commonwealth MycologicalInstitute, ISBN 0851983839, 9780851983837) and Leslie et al., (TheFusarium laboratory manual, 2006, Wiley-Blackwell, ISBN 0813819199,9780813819198), each of which is herein incorporated by reference in itsentirety.

Proteins, including, e.g., certain enzymes, produced by the filamentousfungal species and/or strain(s) can be purified from the filamentousbiomass produced by the organisms. Methods of protein purification areknown to one skilled in the art. Detailed protein purification methodshave been described in Janson and Ryden (Protein purification:principles, high-resolution methods, and applications; Wiley-VCH, 1998,ISBN 0471186260, 9780471186267), Detscher (Guide to proteinpurification, Volume 182 of Methods in enzymology, Gulf ProfessionalPublishing, 1990, ISBN 0121820831, 9780121820831), and Cutler (Proteinpurification protocols, Volume 244 of Methods in molecular biology,Humana Press, 2004 ISBN 1588290670, 9781588290670), which areincorporated by reference in their entireties for all purposes.

Proteins need not be purified from the mats to find utility andusefulness as products. That is, the mat can be processed withoutpurification and be useful; i.e. as a protein source, as a food stuff,and/or as animal feed. The mats can form products unto themselves; themix of biomat produced products in situ are important and valuable.

Lipids of the Fungal Species and/or Strain(s)

As noted above, when cultured in artificial media having a high C:Nratio, filamentous fungi biomats are produced which have a high lipidcontent and a more favorable lipid profile as compared to algae andother lipid producing organisms. The lipids can be extracted from theisolated filamentous biomass. In some cases, the lipids are primarilytriacylglycerides with fatty acid acyl groups. In some instances, thefatty acids are essentially unsaturated fatty acids and/or saturatedfatty acids. The unsaturated fatty acids include oleic acid (18:1),α-linolenic acid (18:3), eicosenoic acid (20:1), and combinationsthereof. Saturated fatty acids include palitic acids (16:0), stearicacids (18:0), arachidic acid (20:0), behenic acid (22:0), andcombinations thereof. Other types of lipids that may be producedinclude, but are not limited to, sterols (e.g. ergosterol, a vitamin inD2 precursor), diacyclyglycerides, carotenoids, saturated fats (e.g.,butyric acid, hexanoic acid, octanoic acid, decanoic acid, dodecanoicacid, tridecanoic acid, tetradecanoic acid, pentadecanoic acid,hexadecanoic acid, heptadecanoic acid, octadecanoic acid, nonadecanoicacid, eicosanoic acid, docosanoic acid, tetracosanoic acid),monounsaturated fats (e.g., tetradecenoic acid, pentadecenoic acid,hexadecenoic acid, heptadecenoic acid, octadecenoic acid, eicosenoicacid, docosenoic acid, cis-tetracosenoic acid), and polyunsaturated fats(e.g., hexadecadienoic acid, linoleic acid, linolenic acid,alpha-linolenic acid, gamma-linolenic acid, parinaric acid,eicosadienoic acid, arachidonic acid, timnodonic acid, brassic acid,clupanodonic acid and docosahexaenoic acid).

The filamentous fungal species and/or strain(s) and/or their progeny arecapable of efficient production of lipids. In some instances, the amountof lipids produced is at least about 1 g/L/day, 5 g/L/day, at leastabout 10 g/L/day, at least about 20 g/L/day, at least about 30 g/L/day,at least about 40 g/L/day, at least about 50 g/L/day, at least about 60g/L/day, at least about 70 g/L/day, or more. For example, the amount ofbiological oil produced is from about 1 g/L/day to about 5 g/L/day, fromabout 5 g/L/day to about 70 g/L/day, from about 10 g/L/day to about 70g/L/day, from about 20 g/L/day to about 70 g/L/day, or from about 30g/L/day to about 70 g/L/day. These values are far greater than thehighest reported value in the literature of about 12 g/L/day (see Dey,P. et al. (2011) Comparative lipid profiling of two endophytic fungalisolates—Colletotrichum sp. and Alternaria sp. having potentialutilities as biodiesel feedstock. Bioresource Technology 102:5815-5823;Gong, Z. et al. (2013) Efficient conversion of biomass into lipids byusing the simultaneous saccharification and enhanced lipid productionprocess. Biotechnology for Biofuels 6:36; Gong, Z. et al. (2014) Lipidproduction from corn stover by the oleaginous yeast Cryptococcuscurvatus. Biotechnology for Biofuels 7:158; Hui, L. et al. (2010) Directmicrobial conversion of wheat straw into lipid by a cellulolytic fungusof Aspergillus oryzae A-4 in solid-state fermentation. BioresourceTechnology 101:7556-7562; Liang, Y. et al. (2014) Microbial lipidproduction from pretreated and hydrolyzed corn fiber. BiotechnolProgress 30:945-951; Liu, C.-Z. et al. (2012) Ionic liquids for biofuelproduction: Opportunities and challenges. Applied Energy 92:406-414;Ruan, Z. et al. (2013) Co-hydrolysis of lignocellulosic biomass formicrobial lipid accumulation. Biotechnol. Bioeng. 110:1039-1049; Sung,M. et al. (2014) Biodiesel production from yeast Cryptococcus sp. usingJerusalem artichoke. Bioresource Technology 155:77-83; Xie, H. et al.(2012) Enzymatic hydrolysates of corn stover pretreated by aN-methylpyrrolidone-ionic liquid solution for microbial lipidproduction. Green Chem. 14:1202-1210).

Lipids can be extracted from the filamentous fungi biomats using variousprocedures. Non-limiting examples of lipid extraction are described inKing et al. (Supercritical Fluid Extraction: Present Status andProspects, 2002, Grasa Asceites, 53, 8-21), Folch et al. (A simplemethod for the isolation and purification of total lipids from animaltissues, 1957, J Biol. Chem., 226, 497-509), Bligh and Dyer (A rapidmethod of total lipid extraction and purification. 1959, Can. J Biochem.Physiol., 37, 911-917), Cabrini et al. (Extraction of lipids andlipophilic antioxidants from fish tissues—a comparison among differentmethods. 1992, Comp. Biochem. Physiol., 101(3), 383-386), Hara et al.(Lipid extraction of tissues with a low toxicity solvent. 1978, Anal.Biochem. 90, 420-426), Lin et al. (Ethyl acetate/ethyl alcohol mixturesas an alternative to Folch reagent for extracting animal lipids. 2004,J. Agric. Food Chem., 52, 4984-4986), Whiteley et al. (Lipidperoxidation in liver tissue specimens stored at subzero temperatures.1992, Cryo-Letters, 13, 83-86), Kramer et al. (A comparison ofprocedures to determine free fatty acids in rat heart. 1978, J. LipidRes., 19, 103-106) and Somashekar et al. (Efficacy of extraction methodsfor lipid and fatty acid composition from fungal cultures, 2001, WorldJournal of Microbiology and Biotechnology, 17(3):317-320).

In another example, lipid can be extracted by methods similar to theFRIOLEX® (Westfalia Separator Industry GmbH, Germany) process is used toextract the biological oils produced by the microorganisms. FRIOLEX® isa water-based physical oil extraction process, whereby raw materialcontaining oil can be used directly for extracting oil without using anyconventional solvent extraction methods. In this process, awater-soluble organic solvent can be used as a process aid and the oilis separated from the raw material broth by density separation usinggravity or centrifugal forces.

After the lipids have been extracted, the lipids can be recovered orseparated from non-lipid components by any suitable means known in theart. For example, low-cost physical and/or mechanical techniques areused to separate the lipid-containing compositions from non-lipidcompositions. If multiple phases or fractions are created by theextraction method used to extract the lipids, where one or more phasesor fractions contain lipids, a method for recovering thelipid-containing phases or fractions can involve physically removing thelipid-containing phases or fractions from the non-lipid phases orfractions, or vice versa. In some cases, a FRIOLEX® type method is usedto extract the lipids produced by the microorganisms and the lipid-richlight phase is then physically separated from the protein-rich heavyphase (such as by skimming off the lipid-rich phase that is on top ofthe protein-rich heavy phase after density separation).

There are at least two stages in the production of lipids by thefilamentous fungal species and/or strain(s): (a) the filamentous fungibiomat accumulation stage and (b) the lipid production stage. Thefilamentous fungi biomat accumulation stage produces a filamentousbiomass, of the fungal species and/or strain(s) such that about 10% toabout 95%, about 20% to about 95%, about 30% to about 95%, about 40% toabout 95%, or about 50% to about 95% of the total filamentous fungibiomat production of the fungal strain is achieved during thefilamentous fungi biomat accumulation stage. In other cases, about 60%to about 95%, about 70% to about 95%, or about 80% to about 95% of thetotal filamentous fungi biomat production of the microorganism isachieved during the filamentous fungi biomat accumulation stage. Inother circumstances, the filamentous fungi biomat accumulation stageproduces filamentous biomass of the microorganism such that at leastabout 10%, at least about 20%, at least about 30%, at least about 40%,at least about 50%, at least about 60%, at least about 70%, at leastabout 80%, at least about 90%, at least about 95%, or 100% of the totalfilamentous biomass production of the microorganism is achieved duringthe filamentous fungi biomat accumulation stage. For example, about 50%to about 95% of the total filamentous fungi biomat production of themicroorganism is achieved during the filamentous fungi biomataccumulation stage.

With respect to the lipid production stage, lipids are producedthroughout all growth stages as they are required for cell growth andproliferation; that is, lipids are produced during the filamentous fungibiomat accumulation stage. While not being bound by theory, it isbelieved that some storage lipids are produced in later stages of biomatgrowth while other storage lipids are produced earlier on during biomatformation. In addition, under low nitrogen conditions the organism willaccumulate storage lipids at a faster rate.

The lipid accumulation stage produces lipids such that about 10% toabout 95%, about 20% to about 95%, about 30% to about 95%, about 40% toabout 95%, or about 50% to about 95% of the total lipid production ofthe microorganism is achieved during the lipid accumulation stage. Insome cases, about 60% to about 95%, about 70% to about 95%, or about 80%to about 95% of the total lipid production of the microorganism isachieved during the lipid accumulation stage. In other circumstances,the lipid accumulation stage produces lipids such that at least about10%, at least about 20%, at least about 30%, at least about 40%, atleast about 50%, at least about 60%, at least about 70%, at least about80%, at least about 90%, or at least about 95% of the total lipidproduction of the microorganism is achieved during the lipidaccumulation stage. Preferably, about 50% to about 95% of the totallipid production of the microorganism is achieved during the lipidaccumulation stage.

Once the lipids are produced in accordance with the present invention,various methods known in the art can be used to transform the biologicaloils into esters of fatty acids for use as ingredients for food orpharmaceutical products. The production of esters of fatty acids cancomprise transesterifying the biological oils produced by themicroorganism. The extraction of the lipids from the microorganisms andthe transesterification of the lipids can be performed simultaneously,in a one-step method. For example, the culture containing the isolatedfungal strain can be exposed to conditions or treatments (or acombination of conditions or treatments) that promote both extraction ofthe lipids and the transesterification of the lipids. Such conditions ortreatments include, but are not limited to, pH, temperature, pressure,the presence of solvents, the presence of water, the presence ofcatalysts or enzymes, the presence of detergents, andphysical/mechanical forces. Two sets of conditions or treatments can becombined to produce a one-step method of extracting and transesterifyingthe lipids, where one set of conditions or treatments favorably promotesextraction of the lipids and the other set of conditions or treatmentsfavorably promotes transesterification of the lipids, so long as the twosets of conditions or treatments can be combined without causingsignificant reduction in the efficiency of either the extraction or thetransesterification of the lipids. Hydrolysis and transesterificationcan be performed directly on whole-cell filamentous biomass.

Alternatively, the extraction of the lipids is performed as a step thatis separate from the step of transesterification of the lipids. Suchtransesterification reactions are performed using acid or basecatalysts. Methods for transesterifying the biological lipids intoesters of fatty acids for use as ingredients for food or pharmaceuticalproducts involves reacting the biological oils containing triglyceridesin the presence of an alcohol and a base to produce esters of the fattyacid residues from the triglycerides.

Alcohols suitable for use in transesterification include any lower alkylalcohol containing from 1 to 6 carbon atoms (i.e., a C₁₋₆ alkyl alcohol,such as methyl, ethyl, isopropyl, butyl, pentyl, hexyl alcohols andisomers thereof). Without being bound by theory, it is believed that theuse of lower alkyl alcohols produces lower alkyl esters of the fattyacid residues. For example, the use of ethanol produces ethyl esters. Ifthe alcohol is methanol or ethanol, the fatty acid esters produced are amethyl ester and an ethyl ester of the fatty acid residue, respectively.Typically, the alcohol comprises from about 5 wt. % to about 70 wt. %,from about 5 wt. % to about 60 wt. %, from about 5% to about 50 wt. %,from about 7 wt. % to about 40 wt. %, from about 9 wt. % to about 30 wt.%, or from about 10 wt. % to about 25 wt. % of the mixture of the lipidscomposition, the alcohol and the base. The composition and the base canbe added to either pure ethanol or pure methanol. In general, the amountof alcohol used may vary with the solubility of the lipids orcomposition containing triglycerides in the alcohol.

The composition comprising triglycerides, the alcohol and the base arereacted together at a temperature and for an amount of time that allowsthe production of an ester from the fatty acid residues and the alcohol.Suitable reaction times and temperatures to produce an ester may bedetermined by one of skill in the art. Without intending to be bound bytheory, the fatty acid residues are believed to be cleaved from theglycerol backbone of the triglyceride and esters of each fatty acidresidue are formed during the step of reacting. The step of reacting thecomposition in the presence of an alcohol and a base is performed at atemperature from about 20° C. to about 140° C., from about 20° C. toabout 120° C., from about 20° C. to about 110° C., from about 20° C. toabout 100° C., or from about 20° C. to about 90° C. Alternatively, thestep of reacting the composition in the presence of an alcohol and abase is performed at a temperature at or greater than about 20° C., 75°C., 80° C., 85° C., 90° C., 95° C., 105° C., or 120° C. Depending on thedesired product, the step of reacting the composition in the presence ofan alcohol and a base is performed for a time from about 2 hours toabout 36 hours, from about 3 hours to about 36 hours, from about 4 hoursto about 36 hours, from about 5 hours to about 36 hours, or from about 6hours to about 36 hours. Instead, the step of reacting the compositionin the presence of an alcohol and a base can be performed for about0.25, 0.5, 1.0, 2.0, 4.0, 5.0, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 10, 12, 16,20, 24, 28, 32, or 36 hours.

The step of reacting the lipids composition, alcohol and base may beconducted by refluxing the components to produce the fatty acid esters,such as PUFA esters. The step of reacting the lipids composition mayalso be carried out at a temperature that does not result in therefluxing of the reaction components. For example, carrying out the stepof reacting the lipids composition under pressures greater thanatmospheric pressure can increase the boiling point of the solventspresent in the reaction mixture. Under such conditions, the reaction canoccur at a temperature at which the solvents would boil at atmosphericpressure, but would not result in the refluxing of the reactioncomponents. Generally, the reaction is conducted at a pressure fromabout 5 to about 20 pounds per square inch (psi); from about 7 to about15 psi; or from about 9 to about 12 psi. Some reactions are conducted ata pressure of 7, 8, 9, 10, 11, or 12 psi. Reactions conducted underpressure may be carried out at the reaction temperatures listed above.Reactions conducted under pressure may be carried out at a temperatureat or greater than about 70° C., 75° C., 80° C., 85° C., or 90° C.

Fatty acid esters are separated from the reaction mixture by distillingthe composition to recover a fraction comprising the ester of the fattyacid. A targeted fraction of the reaction mixture including the fattyacid esters of interest can be separated from the reaction mixture andrecovered. The distillation can be performed under vacuum. Without beingbound by theory, distillation under vacuum allows the distillation to beaccomplished at a lower temperature than in the absence of a vacuum andthus may prevent the degradation of the esters. Typical distillationtemperatures range from about 120° C. to about 170° C., such asperforming the distillation at a temperature of less than about 180° C.,less than about 175° C., less than about 70° C., less than about 165°C., less than about 160° C., less than about 155° C., less than about150° C., less than about 145° C., less than about 140° C., less thanabout 135° C., or less than about 130° C. Typical pressures for vacuumdistillation range from about 0.1 mm Hg to about 10 mm Hg, such as avacuum distillation pressure of at or greater than about 0.1, 0.5, 1,1.5, 2, 2.5, 3, 3.5, or 4 mm Hg.

The lipids extracted from the filamentous fungal species or strainand/or its progeny of the present invention are used to producebiolubricants. As used herein, the term “biolubricants” refers tolubricants produced by using material originated from living or recentlyliving organisms. As used herein, the term “lubricants” refers tosubstances (usually a fluid under operating conditions) introducedbetween two moving surfaces so to reduce the friction and wear betweenthem. Base oils used as motor oils are generally classified by theAmerican Petroleum Institute as being mineral oils (Group I, II, andIII) or synthetic oils (Group IV and V). See American PetroleumInstitute (API) Publication Number 09. One of the single largestapplications for lubricants, in the form of motor oil, is to protect theinternal combustion engines in motor vehicles and powered equipment.Typically, lubricants contain 90% base oil (most often petroleumfractions, called mineral oils) and less than 10% additives. Vegetableoils or synthetic liquids such as hydrogenated polyolefins, esters,silicones, fluorocarbons and many others are sometimes used as baseoils. These are primarily triglyceride esters derived from plants andanimals. For lubricant base oil use the vegetable derived materials arepreferred. Common ones include high oleic canola oil, castor oil, palmoil, sunflower seed oil and rapeseed oil from vegetable, and Tall oilfrom animal sources. Many vegetable oils are often hydrolyzed to yieldthe acids which are subsequently combined selectively to form specialistsynthetic esters.

Thus, the lipids extracted from the filamentous fungi biomats formed bythe filamentous fungal species and/or strain(s) and/or their progeny ofthe present invention can be used to produce ester-based biolubricantcompositions by adding suitable additives. Methods of making ester-basedlubricant compositions are known to one skilled in the art. For anon-limiting example, a quantity of biologically-derived oil comprisingtriglycerides is provided and processed so as to hydrolyze at least someof the triglycerides and form free fatty acids, wherein the fatty acidsare of a type selected from the group consisting of saturated fattyacids, monounsaturated fatty acids, and polyunsaturated fatty acids, andcombinations thereof. The fatty acids are separated by type, such thatat least the monounsaturated fatty acids are substantially isolated fromthe saturated fatty acids and the polyunsaturated fatty acids. Next, atleast some of the monounsaturated fatty acids are modified to form anester product (e.g., comprising triesters), and at least some of thesaturated fatty acids and/or polyunsaturated fatty acids arehydrotreated to yield alkanes (paraffins). Note also that in someembodiments, such ester products can include one or more of thefollowing: mono-, di-, and triester species, and hydroxylated analoguesthereof.

Acid pH Tolerant Enzymes from Filamentous Fungal Species and/orStrain(s)

The genome of Fusarium oxysporum f. sp. lycopersici strain 4287 hasrecently been sequenced and has been shown to carry a variety of genesinvolved in the degradation of lignin, hemicellulose and cellulose.Furthermore, the enzymes involved in the degradation of these materials(e.g., cellulase, xylanase, ligninase, glucuronidase,arabinofuranosidase, arabinogalactanase, ferulic acid esterase, lipase,pectinase, glucomannase, amylase, laminarinase, xyloglucanase,galactanase, glucoamylase, pectate lyase, chitinase,exo-[3-D-glucosaminidase, cellobiose dehydrogenase, and acetylxylanesterase, xylosidase, a-L-arabinofuranosidase, feruloyl esterase,endoglucanase, [3-glucosidase, Mn-peroxidase, and laccase) have beenstudied extensively in F. oxysporum strain F3 (Xiros et al. (2009)Enhanced ethanol production from brewer's spent grain by a Fusariumoxysporum consolidated system. Biotechnol Biofuels 10:4). Consequently,acidophilic filamentous fungal species and/or strain(s) such as Fusariumspecies and the acidophilic filamentous fungal strain designated as MK7,are expected to be fully equipped to hydrolyze complex carbon sources,such as lignocellulosic materials and waste streams (e.g. acid whey). Itis also expected that since these acidophilic filamentous fungal speciesand/or strain(s) are capable of growth at much lower pH (0.7-7.5) incomparison to other filamentous strains (pH>2; Starkey, 1973), theenzymes for lignin, hemicellulose and cellulose degradation will havehigher activities at low pH. Enzymes with high activity under acidicconditions would be especially useful in processes conducted at low pH.

Toxins Produced by Fusarium Species.

Oftentimes, microbial based production requires the use of costly andtime consuming methods to prevent contamination. As discussed above,filamentous acidophilic fungal species and/or strain(s) are highlyresistant to contamination by other organisms. It is known, for example,that members of the Fusarium genus generate toxins (e.g., fumonisins),which have potent antibiotic, insecticidal and phytotoxic properties.Similarly, the filamentous acidophilic MK7 fungal strain requires littleor no external antibiotics to be added when used in the production offilamentous fungi biomats. Some Fusarium species can produce ninedifferent toxins, with production depending on their relationship withdifferent host plants (Marasas et al., 1984, Toxigenic Fusarium species,identity and mycotoxicology, ISBN 0271003480). The production of toxinsalso varies with media used for fermentation. The toxins produced andsecreted by Fusarium species include, but are not limited to, bikaverin,enniatins, fusaric acid, lycomarasmin, moniliformin, oxysporone,trichothecenes, zearelones, various naphthoquinones and anthraquinones(e.g., nonaketide naphthazarin quinones, bikaverin and norbikaverin,heptaketides, nectriafurone, 5-O-methyljavanicin, and anhydrofusarubinlactol).

Additionally, the toxins from Fusarium species may include,4-Acetoxyscirpenediol(4-3-acetoxy-3,15-dihydroxy-12,13-epoxytrichothec-9-ene. A similarcompound, monodeacetylanguidin 4- or 15-acetylscirpentriol),3-Acetyldeoxynivalenol (Deoxynivalenol monoacetate,3″-acetoxy-7″,15-dihydroxy-12,13-epoxytrichothec-9-en-8-one),8-Acetylneosolaniol (Neosolaniol monoacetate,4″,8″,15-triacetoxy-3″-hydroxy-12, 13-epoxytrichothec-9-ene), 4- or15-Acetylscirpentriol (4-Acetoxyscirpenediol), Acetyl T-2 toxin(3″,4″,15-triacetoxy-8″-(3-methylbutyry(oxy)-12,13-epoxytricho-thec-9-ene), Anguidin (Diacetoxyscirpenol), Avenacein,Beauvericin, Butenolide (4-acetamido-4-hydroxy-2-butenoic-acid-lactone),Calonectrin (3″,15-diacetoxy-12,13-epoxytrichothec-9-ene),15-Deacetylcalonectrin (15-De-O-acetylcalonectrin,3″-acetoxy-15-hydroxy-12,13-epoxytrichothec-9-ene), Deoxynivalenol (Rdtoxin, Vomitoxin, 3″, T′, 15-trihydroxy-12,13-epoxytricho-thec-9-en-8-one), Deoxynivalenol diacetate(Diacetyldeoxynivalenol), Deoxynivalenol monoacetate(3-Acetyldeoxynjvalenol), Diacetoxyscirpendiol(7″-Hydroxydiacetoxyscirpenol), Diacetoxyscirpenol (Anguidin, 4,15-diacetoxy-3′-hydroxyl2, 13-epoxytrjchothec-9-ene),Diacetoxyscerpentriol (7″, 8″-Dihydroxydiacetoxyscirpenol),Diacetyldeoxynivalenol (Deoxynivalenol diacetate, 3″,15-diacetoxy-7-hydroxyl2,13-epoxytrichothec-9-en-8-one),Diacetylnivalenol (Nivalenol diacetate, 4, 15-diacetoxy-3′,7′-dihydroxy-12, 13-epoxytrichothec-9-en-8-one), 7″,8″-Dihydroxydiacetoxyscirpenol (Diacetoxyscirpentriol, 4,15-diacetoxy-3″, 7″, 8″-trihydroxy-12, 13-epoxytrichothec-9-ene),Enniatins, Fructigenin, Fumonisin B1 (1,2,3-propanetricarboxylic acid1,-1-[1-(12-amino-4,9,11-trihydroxy-2-methyltridecyl)-2-(1-methylpentyl)-1,2-ethanediyl]ester; macrofusine), Fusarenon (Fusarenon-X, Fusarenon,Monoacetylnivalenol, Nivalenol monoacetate,4-acetoxy-3″,7″,15-trihydroxy-12,13-epoxytrichothec-9-en-8-one) Fusaricacid (Fusarinic acid, 5-butylpicolinic acid), Fusarinic acid (Fusaricacid), F-2 (Zearalenone), HT-2toxin=15-acetoxy-3″,4-dihydroxy-8″-(3-methylbutyryloxy)-12-epoxytricho-thec-9-ene,7″-Hydroxy-diacetoxyscirpenol (Diacetoxyscirpendiol,4,15-diacetoxy-3″,7″-dihydroxy-12,13-epoxytrichothec-9ene),8″-Hydroxydiacetoxyscirpenol (Neosolaniol),1,4-Ipomeadiol(1-(3-furyl)-1,4-pentanediol),1pomeanine(1-(3-furyl)-1,4-pentanetione),1-Ipomeanol(1-(3-furyl)-1-hydroxy-4-pentanone),4-lpomeanol(1-(3-furyl)-4-hydroxy4pentanone), Lateritin, Lycomarasmin,Moniliformin (potassium or sodium salt of1-hydroxycyclobut-1-ene-3,4-dione), Monoacetoxyscirpenol(15-acetoxy-3″,4″-dihydroxy-12,13-epoxytrichothec-9ene),Monoacetylnivalenol (Fusarenon-X), Monodeacetylanguidin(4-Acetoxyscirpenediol), Neosolaniol(8″-Hydroxydiacetoxyscirpeno1,4,15-diacetoxy-3″8″-dihydroxy-12,13-epoxytrichothec-9-ene),Neosolaniolacetate (8-Acetylneosolaniol), Neosolaniol monoacetate(8-Acetylneosolaniol), Nivalenol(3″,4″,7″,15″-tetrahydroxy-12,13-epoxy-trichothec-9-en-8-one), Nivalenoldiacetate (Diacetylnivalenol), Nivalenol monoacetate (Fusarenon-X), NT-1toxin (T-1toxin,4″,8″-diacetoxy-3″,15-dihydroxy-12,13-epoxy-trichothec-9-ene),NT-2 toxin (4″-acetoxy-3″,8″,15-trihydroxy-12,13-epoxytrichothec-9-ene),Rd toxin (Deoxynivalenol), Sambucynin, Scirpentriol(3″,4″,15″-trihydroxy-12,13-epoxytrichothec-9-ene), Solaniol(Neosolaniol), T-1 toxin (NT-1 toxin), T-2 toxin(4″,15″-diacetoxy-3″-hydroxy-8″-(3-methylbutyrlyloxy)-12,13-epoxytrichothec-9-ene),Triacetoxy-scirpendiol(4″,8″,15″-triacetoxy-3″,7″-dihydroxy-12,13-epoxytrichothec-9-ene),Triacetoxy-scirpenol (3″,4″,15″-triacetoxy-12,13-epoxytrichothec-9-ene),Vomitoxin (Deoxynivalenol), Yavanicin, Zearalenol(2,4-dihydroxy-6-(6,10-dihydroxy-trans-1-undecenyl)-benzoicacid-lactone), Zearalenone(6-(10-hydroxy-6-oxo-trans-1-undecenyl)-resorcylic acid lactone). Moredetailed toxins produced by F. oxysporum are described in Tatum et al.(Naphthoquinones produced by Fusarium oxysporum isolated from citrus.1985, Phytochemistry 24:457-459), Tatum et al. (Naphthofurans producedby Fusarium oxysporum isolated from citrus. 1987, Phytochemistry,26:2499-2500), Baker et al. (Novel anthraquinones from stationarycultures of Fusarium oxysporum. 1998, J Ferment Bioeng 85:359-361).Thrane (Fusarium species on their specific profiles of secondarymetabolites, in Fusarium. Mycotoxins, taxonomy and pathogenicity, 1989,ed by Chelkowski J, Elsevier, N.Y., USA, pp 199-225); Baker et al.,Antimicrobial activity of naphthoquinones from Fusaria, Mycopathologia111: 9-15, 1990; Marasas et al. (Toxigenic Fusarium species, identityand mycotoxicology, 1984, Pennsylvania State University Press,University Park, Pa., USA), each of which is incorporated by referent inits entirety for all purposes.

The present invention is further illustrated by the following examplesthat should not be construed as limiting. The contents of allreferences, patents, and published patent applications cited throughoutthis application, as well as the Figures and Sequence Listing, areincorporated herein by reference in their entirety for all purposes.

EXAMPLES Example 1: Strain MK7 in the Natural Environment

Naturally occurring strain MK7 is always associated with algae, archaeaand bacteria in nature and is characterized by average densities of lessthan 0.5 g dry biomass/L spring water (FIG. 1). In addition, MK7 occursin nature as “streamers.” Purcell et al. define “streamers” as follows:“Streamers are submerged aggregations of filamentous and other cellmorphologies projecting into flowing water from a point of attachment”(Purcell et al. (2007) FEMS Microbiology Ecology? 60:456-466). StrainMK7 as a percentage of total streamer biomass is less than 10%.Furthermore, strain MK7 biomass in nature is characterized by greaterthan 30% biomass as macro conidia cells, which are never found insurface fermentation biomats produced by the methods outline in thisdisclosure.

Example 2: Preparation of Artificial Media

MK7-1 liquid medium used in the following procedures was prepared byadding the ingredients listed in Table 1A to deionized water (18.2Mohm), temperature between 22-30° C. followed by adjusting the pH to2.8, pH adjusted lower with 13 N HCl. pH was measured using an OaktonInstruments model 150 pH meter and probe (Orangeburg, N.Y.). The mediumwas then boiled for 20 minutes and allowed to cool to room temperature(˜23° C.) prior to use. Immediately before adding the liquid medium, thepH is checked again using the Oakton Instruments 150 pH meter and probeand adjusted back to pH 2.8 if required.

MK7-3 liquid medium was prepared in the same way using the ingredientslisted in Table 1B.

In some embodiments the carbon source used is not glycerol, but can beselected from various other carbon sources, such as sugars, glycerol,lignocellulosic materials, hydrolysates of lignocellulosic materials,municipal or agricultural waste, food processing waste (e.g. acid whey),industrial waste stream products, potato waste (potato peels, potatodiscards due to degradation, potato cuttings, bruised potato), starchwastes, sugar beet waste, sugar beet pulp, waste from corn processing(i.e. corn steep liquor), wastes from production of biofuels (residuesfrom cellulosic ethanol production, anaerobic digestate, etc.). In thosecases, the media is adjusted to accommodate the contribution ofnutrients from the carbon source used. For example, if the carbon sourceis molasses, acid whey, or lignocellulose, the trace elements neededwill likely be partially or entirely provided by the carbon source andonly the macronutrients may need to be added to the media. In otherembodiments the carbon source may be classified as “food grade.” Inthese instances, there is no expectation of trace elements andmacronutrients associated with the carbon source and so all traceelements and macronutrients must be added.

TABLE 1A Ingredients in MK7-1 medium used for inoculum generation. MK7-1Medium Liquid Plate (g/L) (g/L) Grade Lot # Vendor Location NH4NO3 12.93 ACS 144801A Fisher Waltham, MA Urea 4.3 0.3 ACS A0355726 ACROSSomerville, NJ KH2PO4 10 2 Reagent Eiser- Golden CaCl2•2 H20 2.6 0.4 ACS53H0276 Sigma St. Louis, MO MgSO4•7H2O 2 0.3 Lab 5GJ15040920A FisherWaltham, MA Yeast Extract 2 0 Agar 0 15 Technical 5287994 FisherWaltham, MA Duda Glycerol 75 60 Food 4O19410427A Energy Decatur, ALMicronutrients* mg/L mg/L FeSO4•7 H2O 9.98 4.99 ACS 3562C398 AmrescoSolon, OH ZnSO4•7 H2O 4.4 2.2 USP/FCC 61641 Fisher Waltham, MA MnCl2•4H2O 1.01 0.51 13446-34-9 Fisher Waltham, MA CoCl2•6 H2O 0.32 0.167791-13-1 Fisher Waltham, MA CuSO4•5 H2O 0.31 0.16 Technical 114675Fisher Waltham, MA (NH4)6Mo7O24•4 H2O 0.22 0.11 ACS 68H0004 Sigma St.Louis, MO H3BO3 0.23 0.11 ACS 103289 Fisher Waltham, MA EDTA, free acid78.52 39.3 Electrophoresis 46187 Fisher Waltham, MA pH pH HCl 2.8 4.8ACS 5GK251022 Fisher Waltham, MA

TABLE 1B Ingredients in MK7-3 medium. MK7-3 Medium Liquid (g/L) GradeLot # Vendor Location Urea 8.4 ACS A0355726 ACROS Somerville, NJ KH2PO410 Reagent Eiser- Golden CaCl2•2H20 2.6 ACS 53H0276 Sigma St. Louis, MOMgSO4•7H2O 2 Lab 5GJ15040920A Fisher Waltham, MA Yeast Extract 2 Agar 0Technical 5287994 Fisher Waltham, MA Glycerol 75 Food 4O19410427A DudaDecatur, AL Energy Micronutrients* mg/L FeSO4•7H2O 9.98 ACS 3562C398Amresco Solon, OH ZnSO4•7H2O 4.4 USP/FCC 61641 Fisher Waltham, MAMnCl2•4H2O 1.01 13446-34-9 Fisher Waltham, MA CoCl2•6H2O 0.32 7791-13-1Fisher Waltham, MA CuSO4•5H2O 0.31 Technical 114675 Fisher Waltham, MA(NH4)6Mo7O24•4H2O 0.22 ACS 68H0004 Sigma St. Louis, MO H3BO3 0.23 ACS103289 Fisher Waltham, MA EDTA, free acid 78.52 Electrophoresis 46187Fisher Waltham, MA pH HCl 2.8 ACS 5GK251022 Fisher Waltham, MA

Example 3: Inoculation Process

Cultures to be used for inoculation of trays were grown in 10 Lbioreactors under submerged fermentation conditions in MK7-1 liquidmedium. It should be noted that other bioreactor sizes are amenable andthat the choice of the 10 L reactor size is not to be construed aslimiting. The 10 L reactor was constructed of a 1.3 m long section of10.16 cm diameter clear PVC tubing with a PVC endcap at the bottom. Aplastic aeration port/fitting with a 3 mm orifice was attached to thebottom endcap and tubbing to supply air was attached to the aerationport. A plastic sampling port/valve was attached to the side of theclear PVC wall 15 cm from the bottom of the bottom endcap. The top ofthe clear PVC reactor was covered by sterile gauze, which was held inplace by a loosely fitting PVC endcap with a 3 mm hole to allow gassesto escape from the reactor. The assembled bioreactor is shown in FIG. 2.

Inoculum for the 10 L bioreactor was prepared from an archived culturestock of the filamentous acidophilic MK7 fungal strain (Lot#003). Thearchived stock culture consisted of the filamentous acidophilic MK 7fungal strain mycelial mat grown on a sterile Petri dish containingsolid medium comprised of 1.5% agar (BD Difco granulated agar, Lot#5287994, ThermoFisher, Waltham, Mass.), glycerol and inorganicnutrients as described in Table 1 for MK7-1 plate medium. The agarmedium was prepared by boiling for 20 minutes, allowing the medium tocool to 50° C., and then pouring 25 mL of the solution into a sterilePetri plate. After cooling and solidification, the plate was inoculatedwith a second generation archived freezer stock, by using a sterile loop(heated to red hot in a flame and cooled) to collect a sample from thearchived stock and streaking it on the Petri plate (FIG. 3A).

After 5 days of growth, the mycelial mat completely grew over thesurface of the agar medium (FIG. 3B). The culture was then frozen at−80° C. Five days prior to inoculating the 10 L reactor, a Petri platestock was removed from the freezer and allowing to equilibrate to roomtemperature (23° C.) for 2 hours. The mycelial mat grown on the surfaceof the agar was then removed with sterile forceps (forceps were heatedwith a flame to red hot and cooled with isopropanol) and placed into 350mL of sterile MK7-1 liquid medium in a sterile 1 L glass baffled shakerflask covered with a sterile gauze cloth. The flask was rotated at 200rpm on a VWR OS-500 laboratory shaker (VWR, Radnor, Pa.) for 5 daysprior to its use as inoculum for the 10 L bioreactor.

In preparation for receiving the inoculum, the 10 L bioreactor wassterilized by adding 330 mL of concentrated Na-hypochlorite solution(Chlorox® bleach=8.25% Na-hypochlorite) to 11 L of drinking quality tapwater in the bioreactor and letting it equilibrate for two days. Aftertwo days, another 330 mL of concentrated Na-hypochlorite solution wasadded to the reactor. After one day, the diluted Na-hypochloritesolution was completely drained from the reactor and the reactor wasrinsed with ˜80° C. boiled water by adding 2 L of the hot water andswirling to rinse all of the surfaces inside the bioreactor. The rinsewater was then drained. 3.5 L of sterile MK7-1 liquid medium was addedto the bioreactor and bubbled with sterile air (0.2 um filtered) at arate of 400 mL per minute through the aeration port located at thebottom of the reactor. These bubbling conditions generated bubblesranging in size from 3 to 30 mm in diameter and resulted in mixing andhomogenous distribution of fungal cells throughout the liquid medium(planktonic cells) during growth. Experiments have shown that higherbubbling rates or smaller bubble sizes result in biofilm growth habitthat forms clumps of biomass that stick to surfaces in the bioreactor.Biofilm growth habit therefore is not desirable since a homogeneoussuspension of cells is desirable for inoculation of the tray reactors.The inoculum grown in the 1 L shaker flask was then added to the 10 Lreactor using aseptic technique (spraying all nearby outside surfaceswith 70% isopropanol/30% water prior to opening the top of thebioreactor and not touching any of internal surfaces of the bioreactor).To build additional culture volume in the 10 L reactor, sterile freshMK7-1 liquid medium was added to the reactor after the culture attained6 g/L dry filamentous biomass density. If fresh MK7-1 liquid medium isto be added to the reactor, the volume should be no more than 9 timesthe liquid culture volume in the reactor. Dry filamentous biomass wasmeasured by collecting a sample via a side port on the bioreactor whilethe aeration system is operating, and filtering a known volume through a0.22 um filter (Millipore, Cat# GSWP04700, Darmstat, Germany) using avacuum filtration apparatus (Millipore, Cat# WP6111560, XX1004700,XX1004705, Darmstat, Germany). The pre-weighed filter, plus the wetfilamentous biomass is dried at 50° C. for 4 h in the BenchmarkScientific Incu-Shaker Mini (Edison, N.J.) and then weighed on a MettlerToledo scale model MS3035 (Columbus, Ohio).

The filamentous acidophilic MK7 fungal strain has a specific growth rateof about 0.024 h⁻¹ in the 10 L reactor (FIG. 4) and growth during theexponential phase will follow the equation:

x=x _(o) exp^(μt)  (Eqn 1)

Where x is the final biomass, x_(o) is the initial biomass, μ is thespecific growth rate, and t is the time. For use as inoculum in the trayreactors, the culture cell density in the 10 L reactor should be above 6g/L dry weight and in the late exponential growth phase (FIG. 4;exponential growth is the period of growth in a culture when cellnumbers are continuously doubling; late exponential growth is the periodjust prior to cessation of exponential growth when cell growth ratesbegin to decline). If culture medium with lower cell densities is usedfor inoculum, it will result in significantly slower biomat formation(lag phase that is longer than 2 days) and is thus not desirable.

Inoculum is defined herein as essentially composed of planktonic cells,which are defined as single cells that are not clumped or aggregatedtogether and are about 4 microns in width to 5-20 microns in length.

To inoculate surface tray reactors for surface fermentation or solidsubstrate surface fermentation, the liquid culture was removed from the10 L reactor via a port near the bottom of the reactor whilecontinuously being mixed by bubbling. The culture to be used forinoculum was removed in an aseptic manner by spraying the inside of theport with 70% isopropanol/30% deionized water mixture, then opening thevalve to allow about 25 mL of culture to be expelled and thus rinse thevalve. This waste inoculum culture was disposed. The inoculum culture isadded directly to the tray reactor medium as described in Example 3.

Example 4: Growth of Strain MK7 in Tray Reactors Via SurfaceFermentation

Filamentous acidophilic MK 7 fungal strain, were grown in shallow trayreactors. It should be noted that different tray sizes are amenable tothe teachings of this innovation. In this example, the inside dimensionsof the polyethylene trays were 41.27 cm wide by 61.28 cm long with 2.54cm tall sidewalls (total surface area available for mat growth=0.253 m²;FIG. 5; Winco, Idaho Falls, Id.). It is desirable that trays be clean ofdebris and chemicals as well as sterilized prior to use to minimizepotential contamination. Consequently, prior to use, the trays werethoroughly washed with soap and warm drinking quality tap water (50-70C), thoroughly rinsed with the warm tap water for 1 minute, and removalof all soap residues was validated. This was followed by spraying allsurfaces of the trays until all surfaces were wetted with a solution of70% isopropanol/30% deionized water (18.2 Mohm) and wiping the trayswith gloved hands using a paper towel soaked in the alcohol mixture.Trays were then placed inside a rack system such that said tray canaccept and hold liquid medium (described below) without spilling andallowed to dry.

The tray and rack system for used for the surface fermentation processdescribed here provides all the necessary components to form a biomatand enable rapid growth of the biomat. The rack system used to hold thereactor trays was a chrome-coated steel, 39 tray rack purchased fromGlobal Equipment Company (Chicago, Ill.; FIG. 5A, B). Clear plasticSaran®-like wrap 16 inches wide (Costco, Bozeman, Mont.) was used towrap and enclose the rack system, and isolate the trays from thesurrounding room. This enabled control of environmental conditions(humidity, air flow) and minimized contamination (FIG. 5A, B).Humidified sterile air was blown into the enclosed rack at a rate of 800mL/minute via bubbling through 200 mL of deionized water (18.2 Mohm),water temperature 22-30° C. (to humidify the air) and passage through anautoclaved 0.2 um filter (Millipore, Cat# SLFG85000, Darmstat, Germany)to remove microorganisms. Ideally, the rate of airflow is such that itcreates a slight positive pressure in the rack system (>0.1 psi) therebyminimizing the amount of airborne contaminants from entering theenclosed tray and rack system until the biomat reaches the desireddensity and/or consistency.

While the microbial mat is active, cells are respiring; that is,producing carbon dioxide and heat, as well as consuming oxygen.Accumulating carbon dioxide can reduce availability of oxygen and shouldbe limited. Thus, airflow should be such that it flushes out carbondioxide that is produced and accumulated during microbial respiration.Additionally, airflow should be such that it removes excess heatgenerated during microbial respiration and supplies adequate oxygen tothe respiring cells. Airflow should be adjusted to meet these needs. Forexample, as need increases when a greater number of trays are used,airflow should be increased to meet the increased temperature andatmospheric needs. Airflow should not be strong enough to perturb thefungal hyphae and inhibit their growth and function. Ideally, in a traysystem airflow would result in air flow across the trays. In oneembodiment, the airflow can be generated by a fan that passes airthrough a 0.2 um filter and across the mats. The fan speed and resultingair flow can be controlled by a smart sensor/actuation system based upontemperature, carbon dioxide and oxygen sensors positioned in the rack.

Temperature of the tray system ranged from 25°±2° C. during growth.Temperature was measured using ThermoScientific Genesys 10S Series,Biomat 3S, Evolution 60S software and sensor system (Thermo Fisher.Waltham, Mass.). The thermocouple sensors were placed 20 mm inside thetray rack system at mid-height and at the top of the tray rack system.

MK7-1 medium was prepared as described in Example 2 [addition ofnutrients, pH adjustment, boiling and cooling to room temperature (˜23°C.)]. After medium preparation, inoculum culture from the inoculumreactor was obtained (see Example 3) and added at a rate of 7.5% (volumeto volume) of the MK7-1 medium in a pot. For example, 113 mL of inoculumwas added to 1.5 L of medium in the pot. This ratio of inoculum tomedium provides adequate conditions for rapid growth of the cells andmat formation. In this embodiment, the desired dry cellular filamentousbiomass after inoculation of the fresh medium is 0.45 to 0.75 g/L.However, densities ranging from 0.01 to 100 g/L could potentially beused to successfully generate a biomat in the present tray system.Reducing the inoculum to medium ratio results in slower growth asdescribed in Example 3.

The volume of carbon substrate to the volume of medium impacts theresulting rate of biomass production. In general, when theaforementioned ratio is too small, the growth rates slow down due tolack of available carbon; that is, they become carbon limited. When theaforementioned ratio is too large, the resulting osmotic pressurebecomes too great and the biomass growth rates diminish. Furthermore,when carbon is limited, resulting biomass density and biomasscohesiveness is small, thereby diminishing the processing and handlingadvantages offered by the surface fermentation process. For example, thefilamentous fungal strain designated as MK7 has optimal growthconditions when the aforementioned ratio is between 8-15%.

The medium/cell suspension was mixed with a sterilized large plasticspoon (30 cm long, sterilized by rinsing with the alcohol mixture) and1.5 L of the resulting mixture was added to each tray using a sterilized(rinsed with the alcohol mixture) graduated cylinder. After all trayswere loaded into the rack system, the rack system was wrapped with theclear plastic.

After 6 days of incubation, the resultant biomats were 3 to 10 mm thickwith enough tensile strength and structural integrity so that they canbe handled without tearing (FIG. 6). The biomats were harvested by firstremoving the clear plastic wrap around the rack system and removing thetrays from the rack. Biomats were removed from the trays by hand, placedin a 12.7×23 cm Pyrex glass tray and gently rinsed for two minutes withdrinking quality tap water. The rinsed biomats were either left on theglass tray and dried or placed in a 3.7 L plastic bag and frozen. Todry, the biomats were placed in a temperature controlled oven and heatedat 60°±1° C. for 45 minutes to deactivate many of the enzymes and limitbiochemical transformations within the mat, followed by heating at50°±1° C. until the dry weight did not change (approximately 48-72hours). Average dry weights of biomats produced given the aboveconditions were 81 g dry filamentous biomass per tray for thefilamentous acidophilic MK7 fungal strain. This is equivalent to 54 g/L,324 g/m² surface area and a productivity of 0.37 g/L/hour. Averagemoisture content of the undried biomat was 0.17 dry filamentousbiomass/g or 83% liquid.

Cellular growth in trays typically occurs according to the growth curveshown in FIG. 7. Cells grow in a planktonic (homogenous/evenlydistributed cells throughout the medium) state until about 48 hours ofgrowth. After 48 hours, the cells aggregate at the surface of the mediumand begin to form a biofilm; in other words, a microbial mat where cellsare intertwined and stuck together. The mat is a very thin skin atfirst, but continues to grow rapidly until some limiting factor such aslack carbon substrate or other nutrient limits growth.

Due to the sensitivity of the biomat to disturbance and consequentdecline of growth, it is important that trays remain undisturbed and theintegrity of the biomat is maintained during the entire growth period.Disturbances that impact the biomat include excessive shaking of thetray, applying pressure to the mat, applying liquid to the surface ofthe biomat, rapid air flow across the biomat, disturbing, breaking orcompression of the hyphae, or physical disruption of the mat itself.These types of disturbances result in loss of the advantages of growinga biomat, which include rapid growth rates and high filamentous biomassaccumulation per liquid volume and surface area.

It is hypothesized that the aerial hyphae and mycelia play an importantrole in supplying oxygen for respiration of cells in the entire biomat.Thus, the formation, growth and function of aerial hyphae/mycelia isimportant for rapid growth of the biomat. Consequently, any disturbancethat impacts formation or growth of these hyphae/mycelia results in adecline of biomat growth.

The surface fermentation method and medium described above provides allthe necessary components to form a biomat and enable rapid growth. Awide variety of tray systems were tested using the above concepts andnearly equivalent productivity per unit area was obtained. For example,using 7.5% glycerol and a 10:1 C:N ratio, productivities remainedconstant at about 44 g/m²/d during the growth phase as the surface areasof the bioreactors were increased from 0.02 to 1 m².

Example 5: Effects of Tray Size on Filamentous Fungi Biomat Growth andProductivity

To test the effects of tray size on biomat growth and productivity,strain MK7 filamentous fungi biomats were grown on MK7-1 medium with7.5% glycerol in 0.02 m² Pyrex® glass trays, 0.25 m² polypropylene traysand a 1.77 m² plastic lined tray. Liquid medium volume to surface arearatios were 6 L/m² for all treatments. Growth rates were only minimallyaffected by tray size and linear growth rates were observed after 6 days(FIG. 8). Dry biomass productivity was 1.32, 1.54, and 1.57/m²/h for the0.02, 0.25, and 1.77 m² trays, respectively.

Example 6: Growth of Strain MK7 on Different Media

The growth characteristics of filamentous acidophilic MK7 fungal straingrowth characteristics (i.e. growth rate, cell density, substrateconversion efficiency, mat formation) vary dramatically as a function ofchoice of growth medium and whether the cultures are grown inSSF/submerged fermentation or SSSF/surface fermentation conditions. Thefilamentous acidophilic MK7 fungal strain was cultivated on a historical(May 2009 to November 2012) medium MK7A, initially designed to mimic thechemistry found in the organism's natural environment in YellowstoneNational Park but with increased nitrogen, phosphorous, calcium andmagnesium concentrations to match a nutrient source found beneficial forother filamentous fungi (Table 2; MK7A). MK7-1 medium was developed toenhance and improve the filamentous acidophilic MK7 fungal strain growthcharacteristics, especially in regards to mat formation by surfacefermentation (Table 2). Specifically, phosphate, calcium and magnesiumand nitrogen were increased and an additional nitrogen source was added(urea). Increasing calcium, magnesium and adding urea specificallyincreased growth rates and enhanced mat formation.

TABLE 2 Chemical components of historical medium MK7A (submergedfermentation) compared to Modified MK7-1 designed for high-densityfilamentous biomass formation by surface fermentation. MK7A MediumModified MK7-1 Medium g/L g/L NH4NO3 3.5 NH4NO3 12.9 KH2PO4 2.0 Urea 4.3CaCl2•2H2O 0.4 KH2PO4 10.0 MgSO4•7H2O 0.3 CaCl2 2.0 MnSO4•7H2O 0.5MgSO4•7H2O 2.0 Micronutrients** mg/L Micronutrients* mg/L FeCl3•6H2O20.00 FeSO4•7H2O 4.99 ZnSO4•7H2O 0.22 ZnSO4•7H2O 2.20 CoSO4 0.01MnCl2•4H2O 0.51 CuCl2•2H2O 0.05 CoCl2•6H2O 0.16 NaMoO4•2H2O 0.03CuSO4•5H2O 0.16 Na2B4O7•10H2O 4.50 (NH4)6Mo7O24•4H2O 0.11 VOSO4•2H2O0.03 H3BO3 0.11 Glucose g/L 40.0 EDTA, free acid 39.3 pH 2.5 Glucose g/L125.0 C:N ratio 16.3 pH 2.8 C:N ratio 7.5

Initial experiments with MK7A medium were done exclusively undersubmerged fermentation conditions in shaker flasks. Maximum growthrates, conversion efficiency (g of carbon substrate converted tofilamentous biomass) and highest filamentous biomass produced underthese conditions were 0.072 g/L/h; 22%; and 8.6 g/L, respectively (Table3).

TABLE 3 Growth characteristics of the filamentous acidophilic MK7 fungalstrain under a variety of conditions. Average Maximum Maximum BiomassMaximum Growth Biomass Biomass production biomass Rate Produced Producedrate Produced Conversion Biorector Carbon source (g/L/hr) g/Lg/m{circumflex over ( )}2 g/m{circumflex over ( )}2/day (days)Efficiency Submerged - aerated 4% glucose minimal 0.072 8.6 5 22%Submerged - aerated 4% glucose MK7-1 0.126 15.1 5 38% Submerged -aerated 4% Glycerol MK7-1 0.28 24.1 23 31% 0.02 m{circumflex over ( )}2Tray - 100 ml 4% glucose MK7-1 0.135 19.5 110 18.3 6 49% 0.02m{circumflex over ( )}2 Tray - 100 ml 12.5% Glycerol MK7-1 0.44 64.7 32452.9 6 52% 0.25 m{circumflex over ( )}2 Tray - 1.25 L 12.5% GlycerolMK7-1 0.46 66.2 331 55.2 6 53%

To increase cell density, MK7A medium was utilized in conjunction withsurface fermentation conditions. Biomats formed in carbon concentrationsbetween 4-15% with glucose or sucrose and 4-30% with glycerol. Urea wasfound to increase rates of production in surface fermentation conditionsand NH₄NO₃, NH₄PO₄, NH₄SO₄ are alternative sources of NH₄. Addition ofurea has benefits of less expensive market prices as compared to otherNH₄ sources. 12.5% carbon substrate was found to be ideal for increasingfilamentous biomass densities up to 180 g/L (density after biomatremoval from tray) and optimizing growth rates.

High density biomats produced with surface fermentation and MK7-1 mediumhave a number of advantages over submerged fermentation conditions (witheither media) including: (1) increased filamentous biomass density up to180 g/L (density after biomat removal from tray) compared to maximum of24.1 g/L with submerged fermentation (Table 3), (2) increased growthrates up to 0.46 g/L/h compared to maximum rates of 0.28 g/L/hr undersubmerged conditions, (3) increased density of carbon feedstock from 7.5to 12.5% for maximum growth rates, (4) much easier harvesting conditionsfor filamentous biomass especially when high density filamentous biomassis produced (e.g. centrifugation not necessary), (5) there is no need toaerate the filamentous biomass as compared to large complex aeratedbioreactors for submerged fermentation (6) much more scalable andexpandable to use surface tray systems compared to very large commercialsubmerged fermenters (7) less liquid waste.

Decreasing C:N ratios to <10:1 were also highly beneficial forfilamentous biomass production and increased the levels of proteinversus lipids. Historically, MK7A medium was designed for production oflipids by the filamentous acidophilic MK7 fungal strain especially atC:N ratios above 30:1. Varying C:N ratios along with culture conditionsallow for the tailoring of lipid concentrations in the filamentousacidophilic MK7 fungal strain biomass between 5 to up to 60% of weightof biomass. Fatty acid profiles for filamentous biomass were verysimilar between MK7A and MK7-1 media with various simple carbonsubstrates (e.g. glycerol, glucose, sucrose), but temperature was foundto increase the concentration of polyunsaturated fatty acids with theomega-3 linolenic acid increasing over time at lower relativetemperatures (FIG. 9).

Example 7. Growth of Strain MK7 on MK7A and MK7-1 Media

Strain MK7 filamentous fungi biomats were produced under surfacefermentation conditions using MK7A and MK7-1 media with either sucroseor glycerol as a carbon source (feedstock). To evaluate filamentousfungi biomats produced from MK7A and MK7-1 media, five different mediaformulations were prepared: 1) 4% sucrose in MK7A medium, 2) 4% sucrosein MK7-1 medium, 3) 4% glycerol in MK7A medium, 4) 10% glycerol in MK7-1medium, and 5) 12.5% glycerol in MK7-1 medium. The pH of all five mediaformulations was adjusted to 2.7 using appropriate additions ofconcentrated HCl followed by boiling for 10 minutes. After cooling toroom temperature (˜23° C.), a 7.5% volume/volume of strain MK7 inoculumin exponential growth phase was added to each media. The pH wasreadjusted to 2.7 and 250 mL aliquots of the inoculated media were addedto sanitized 0.023 m² Pyrex® glass trays. The trays were then placed ina tray rack system and the mixtures of media with inoculum were allowedto incubate at 23°±1° C.

Based on results from previous experiments, it is expected that biomasswill form on all of the media combinations. It is also expected thatbiomass will form more quickly on MK7A media relative to MK7-1 media.This is due to the more hospitable chemical conditions of the MK7A mediafor growth (e.g. lower ionic strength/osmotic pressure, lower ammoniumconcentration). In time however, filamentous fungi biomats that developon the surface of the MK7-1 media will grow with a faster growth ratethan biomass growing on MK7A media. That is, both systems have differentgrowth rate curves with the MK7-1 media exhibiting fast growth in theearly stages followed by reduced growth rates in the later stages.Conversely, filamentous fungi biomats grown on the MK7-1 media haveinitially relatively slow growth rates in the early growth stagesfollowed by extremely rapid growth rates in the later stages of biomassgrowth. Ultimately, filamentous fungi biomats grown on the MK7-1 mediabecome thicker and have greater tensile strength than biomats grown onMK7A media. It is expected that the significantly lower concentrationsof nutrients in the MK7A media (e.g. N, P, K) will result in earlynutrient limitation, causing growth inhibition with biomats that are notas thick or strong as biomats produced on MK7-1 media.

Example 8: Structure of Biomats Produced by Strain MK7

The structure of the biomat was determined by transmitted lightmicroscopy. Here, biomats were produced from strain MK7 grown for 5 dayson MK7-1 medium with 7.5% glycerol. Biomats were harvested, frozen (−20°C.), and dissected into 1 cm×1 cm square blocks before embedding in 10%gelatin (Sigma G2500-3000 Bloom gelatin, Sigma-Aldrich, Saint Louis,Mo.) in cryomolds (VWR 25608-916, Radnor, Pa.). The gelatin/tissuesample was flash-frozen by exposing the cryomolds to the vapor phase ofa liquid nitrogen bath before placing at −20° C. overnight.

Cyrosectioning was accomplished using a Leica model 39475214Cryosectioner (Leica, Wetzlar, Germany). Samples were removed from thecryomolds and coated with OTC tissue freezing medium (Leica 14020108926)prior to cryosectioning into 10-50 μm thick slices. Sample slices werevisualized and imaged using a transmitted light microscopy (Microscope:Zeiss AxioObserver; Camera: Zeiss AxioCam HRc, Carl Zeiss, Oberkochen,Germany).

With glycerol mats, at least two distinct layers were observed: (a) adense bottom layer and (b) an aerial hyphae layer. In some samples atleast three structurally different layers were visible: (a) a densebottom layer, (b) an aerial hyphae layer and (c) a transition zone layer(see FIGS. 10A and B). Typically, the aerial hyphae layer is mostvisibly dominant, followed by the dense bottom layer, while thetransition zone layer, when present and/or visible, is smallest. In someinstances, for example the biomat shown in FIG. 10A, the visible ratioof the (a) dense bottom layer to the (b) aerial hyphae layer to the (c)transition zone layer was about 3.86 to about 9.43 to about 1. Inanother sample, such as the biomat shown in FIG. 18B, the ratio wasabout 1.7 to about 3.7 to about 1. There was no visibly distincttransition zone layer apparent in FIG. 18C.

Additional optical imaging of an MK7-3 grown biomat indicated no lipidsor pigments present in the top aerial hyphae layer as compared to thedense bottom layer (FIGS. 11A and 11B). Aerial hyphae are foundextending from the mat and each is exposed to the atmosphere withoutliquid between the hyphae. This distinguishes them from hyphae/myceliain the other layer(s) of the mat, which are separated by liquid and/oran extracellular polysaccharide/protein matrix. Aerial hyphae areresponsible for oxygen transfer and CO2 transfer. Oxygen accessibilityresults in oxygen absorbing hyphae in the top aerial hyphae layer. Theseaerial hyphae appear to be longer than the hyphae/mycelia found in thelower mat layer(s) (compare FIG. 11B with FIG. 11C). The aerial hyphaeof the top layer also tend to have a preponderance of verticalorientation, that is they tend to be oriented perpendicular to thefilamentous fungi biomat air interface.

Vertical orientation was not as predominant in the hyphae of the densebottom layer (FIG. 11C). Here, the hyphae tend to be intertwined andhave a mix of orientations and have a preponderance of horizontalorientations. The hyphae of the bottom layer also appeared to contain apurple pigment, which was not evident in the top aerial layer.Preliminary experiments indicated that the bottom layer containsapproximately 30% lipids and that the hyphae are embedded in a proteinand/or polysaccharide matrix. The bottom layer hyphae are also theprimary storage area for pigments, lipids, carbohydrates, and protein.

Biomats produced from strain MK7 grown on (1) MK7-1 medium and (2) MK7-3medium, were harvested after 5 days and frozen at 20° C. The thicknessof both biomats ranged from 2 to 4 mm. One cm² square sections were cutfrom the two frozen biomats in triplicate and then cut in halflatitudinally, producing top and bottom half cross sections, eachapproximately 1.5 mm thick. \

The average density for the biomat grown in MK7-1 media was 0.131 g/cm³for the top latitudinal section (standard deviation=0.068) and 0.311g/cm³ for the bottom latitudinal section (standard deviation=0.032). Theratio of top to bottom densities was 0.42.

For the biomat grown in MK7-3 medium, the average density for the toplatitudinal section was 0.102 g/cm³ (standard deviation=0.048) while theaverage density for the bottom latitudinal section was 0.256 g/cm³(standard deviation=0.010. The ratio of top to bottom densities was0.40.

Example 9: Tensile Strength of Biomat Produced by Strain MK7

The tensile strength of an MK7 biomat grown for 5 days on MK7-3 mediawas evaluated. Here, a 25.4 cm wide, 46 cm long, 3.5 mm thick mat wasused. The water content of the mat was about 85%, equating toapproximately 15% dry weight. The total dry weight was 70 g/0.25 m² trayor 280 g/m². The mat had a density of 0.08 g/cm³.

To measure tensile strength, one end of the mat was clamped into astationary position while the other end was clamped to a free movingapparatus. The free moving apparatus was itself attached to a scale thatmeasures applied tension. A steady and slow tension was applied to themat by pulling on the scale over several seconds until the mat broke.The measured tension required to break/rip/tear the mat ranged from 0.28kg/2.54 cm of mat width to 0.58 kg/2.54 cm of mat width, which isequivalent to 0.11 kg/cm of mat width to 0.23 kg/cm of mat width. Theaverage was 0.5 kg/2.54 cm mat width, or 0.2 kg/cm mat width.

Example 10: Growth of Strain MK7 Biomats on Crude Glycerin

Dense strain MK7 biomats were produced in 8 days using crude glycerin asa carbon and nutrient source (feedstock). Crude glycerin, a by-productof biodiesel production, was obtained from W-2 Fuels (Product CodeGL32000, Batch 4300, CAS No. 56-81-5, Adrian, Mich.). The crude glycerinwas comprised of 75-85% glycerin, 2-10% water, 2-5% salts, 1-2% fats,oils or esters, and <1% methanol.

A 7.5% concentration of crude glycerin in drinking quality tap water(weight:volume) was supplemented with either full-strength or ½ strengthMK7-1 medium salts to create 11 L of full strength and ½ strength MK7-1medium. The pH of these solutions was adjusted to 4.8 followed byboiling for 10 minutes. After cooling to room temperature (˜23° C.), a5% volume:volume strain MK7 inoculum prepared as described in Example 3was added to the medium. The pH was readjusted to 4.8 and 1.5 L aliquotsof the inoculated crude glycerin medium were added to sanitizedpolypropylene 0.25 m² trays before placing the trays in a rack system.

The mixtures were incubated at 23°±1° C. and resulted in flexible,relatively dense biomats that were about 4 mm thick after 8 days atwhich time they were harvested. Biomats were dried at 50° C. for 72 hand the average dry weights ±standard deviations were 30.3±3.1 g (n=6)for the full strength media treatment and 30.2±2.8 g (n=8) for the ½strength media treatment. The average conversion of glycerin to drybiomat was 34% and the density of the moist mats on a dry biomass weightbasis was 0.03 g/cm² for both treatments.

Example 11: Hyphael/Mycelial Structure of Strain MK7 Biomats Grown onWheat Distillers Solubles

Consolidated strain MK7 filamentous fungi biomats were produced in 7days using dried wheat distillers solubles (ds) as the carbon andnutrient source (feedstock). The wheat ds were comprised of 31.5%protein, 8.6% oil, 2.8% starch, 13.5% sugar, 2.7% fiber, 8.5% ash, 0.19%calcium, 0.29% magnesium, 1.7% potassium, 0.78% phosphorus, and 3.5%sulfate. Two growth media treatments were prepared: Treatment 1 used 5%ds dry weight in water and Treatment 2 used 5% ds dry weight in ½strength MK7-1 salts medium. The pH of the mixtures was adjusted to 3.4.The mixtures were inoculated with 7.5% (volume:volume) of strain MK7inoculum prepared as described in Example 3 and 175 ml of that mediumwas added to alcohol sterilized 12.7×12.7 cm plastic trays. Filamentousfungi biomats were harvested after 7 days of incubation at roomtemperature (˜23° C.). Biomats grown on 5% ds as the sole carbon andnutrient source (Treatment 1, without MK7-1 salts) were an average of2.7 mm thick (n=3 trays), showed no distinct layering, and had anaverage dry weight of 0.83 g with an average density of 0.019 g/cm³ anda conversion efficiency of approximately 10%. No aerial hyphae wereobserved and the mats were saturated with liquid throughout; i.e. thetop surface of the mats were at the surface of the liquid.

Biomats grown on 5% ds supplemented with MK7-1 salts (Treatment 2) werean average of 6.4 mm thick and had an average dry weight of 3.11 g withan average density of 0.030 g/cm³ and a conversi8on efficiency ofapproximately 40%. These mats developed an extensive fluffy white aerialhyphae system that was about 4-7 mm thick immediately above a distinctdenser layer that was about 0.9 mm thick. The density of the upper layerwas 0.011 g/cm³ and the density of the lower layer was 0.148 g/cm³.

Example 12: Growth of Strain MK7 Filamentous Fungi Biomats on Corn SteepLiquor and Corn Steep Liquor/Starch as the Carbon and Nutrient Source

Dense strain MK7 filamentous fungi biomats were produced in as little as4 days using corn steep liquor as the sole carbon and nutrient source(feedstock). Further, corn steep liquor with 5% starch addition was alsoshown to be capable of producing dense filamentous fungi biomats. Cornsteep liquor is a viscous by-product of corn wet-milling and has acomposition of amino acids, vitamins, and minerals that makes itsuitable as a supplement for microbial fermentations. The corn steepliquor used in this example was purchased from Santa Cruz Biotechnology,Inc. (Dallas, Tex.; Lot# B0116). These experiments demonstrated the useof corn steep liquor as a replacement for MK7-1 nutrients. Treatmentsincluded 10% and 20% corn steep liquor as the sole carbon and nutrientsource, and 10% and 20% corn steep liquor plus 5% starch (each) with thestarch providing additional carbon for filamentous fungi biomat growth.

Four batches of media were prepared by adding 10% or 20% corn steepliquor to 1 L volumes containing 0 or 50 g dry starch. The media wasadjusted to pH 3.6 by adding a suitable amount of HCl and boiled for 15minutes in a suitable container. After cooling to room temperature, thepH of the mixture was readjusted to 3.6 and the mixture inoculated with7.5% strain MK7 inoculum as prepared in Example 3. Aliquots of 175 ml ofmedia were added to five square trays (0.016 m² surface area/tray) andthe prays incubated in a rack system at 23°±1° C. Filamentous fungibiomats were harvested after 6 days. The average final pH for the cornsteep liquor treatments at 10%, 20%, 10%+starch, and 20%+starch were3.97, 3.69, 4.23, and 4.15, respectively. The average biomassweight±standard deviation for these treatments were 1.1±0.2, 0.1±0.1,2.3±0.1 and 2.1±0.2 g, respectively.

Example 13: Conversion of Cattle Feedlot Lagoon Water to FilamentousFungi Biomats

Growth experiments were conducted using cattle feedlot lagoon water asthe sole carbon and nutrient source. The initial dissolved organiccarbon content of the waters was 4 g/L and initial total dissolvednitrogen content was 0.8 g/L.

Feedlot lagoon water was adjusted to pH 2.6 with concentrated HCl andinoculated with 7% strain MK7 inoculum as prepared in Example 3.Sterilized 0.25 m2 polypropylene trays were filled with 1.5 L of theinoculated waste water and placed into a tray rack system for incubationat 24°±1° C. Filamentous fungi biomats began to form on the surface ofthe liquid 2 days after inoculation. After ten days, the filamentousfungi biomats and remaining liquid were collected in a single vessel anddried prior to analysis for total C and N using a Costech total C and Nanalyzer (ECS 4010, Costech Analytical Technologies, Valencia, Calif.).The average dry biomass produced per tray was 6.531 g (n=2). Analyses ofthe mats and residual liquid revealed that about 77% of the carbon and99-100% of the nitrogen was removed from the feedlot lagoon wastewaterby the mats (method detection limit ˜1%). Carbon and nitrogen removalrates for the system were 6.8 and 1.2 mg/L/h when averaged across the10-day period. According to present understanding, it is possible toremove most of the C and nearly all of the N from feedlot waters byacidifying the lagoon to pH 2.6 with HCl and inoculating with strainMK7. It is further possible to treat lagoons directly, resulting in afloating filamentous fungi biomat on the feedlot water on site that canthen be harvested for subsequent use.

Example 14: Growth of Strain MK7 Biomats on Acid Whey Surrogate Medium

Dense strain MK7 biomats were produced from Acid Whey Surrogate Medium(AWS) in 7 days. Composition of the AWS medium was based on the typicalcomposition of acid whey as described in Tsakali et al. (2010).Composition of the Tsakali medium and the AWS Medium used in thisExample is described in Table 4.

Biomats were produced in sterilized 12.7×12.7 cm (0.016 m²)polypropylene trays using 87 mL of pH 4.8 AWS medium. The medium wasprepared by mixing the ingredients listed in Table 4 in a 1 L Erlenmeyerflask, adjusting the pH to 4.8, and then boiling for 10 minutes. Afterthe medium cooled to about 23° C., a 7.5% volume (volume/volume) ofinoculum in exponential growth phase was added to the flask. Thisinoculum was generated as described in Example 3. Aliquots of 87 mL ofthe inoculated culture medium were added to isopropyl swabbed trays(Example 3) and the trays were placed on a larger (0.25 m²) tray mountedin the tray rack system as described in Example 3. The cultures wereallowed to incubate at 25±1° C. resulting in relatively dense biomatsthat were harvested after 7 days (FIG. 18). The mean pH value of theresidual liquid after 7 days was 6.9±0.1. Thus, the growth processneutralized the pH of AWS from 4.8, typical of acid whey, to nearneutral (˜7). Transmitted light microscopy revealed the filamentousnature of the biomats generated on AWS medium (FIG. 18). The averagethickness of the moist biomats was 4±0.5 mm. Biomats were dried at 50°C. for 30 h and the resulting average dry weights were 1.88±0.2 g. Theaverage density of the moist biomats was 0.29 g/cm³. The averageconversion of dry feedstock (lactose and total proteins) to dry biomatwas 42.2%.

TABLE 4 Composition of a typical acid whey as described in Tsakali etal., 2010, and the Acid Whey Surrogate medium (AWS) used in this Examplefor growing MK7 biomat. Tsakali AWS (%) (%) Water 94.5 94.5 Dry Matter5.5 5.5 Lactose 4 4 Lactic 0.4 0 Acid Total Protein* 1 1 Citric Acid 0.10.1 Minerals** 0.6 1.3 pH 4.8 4.8 *Total protein source for AWS was wheyprotein concentrate as 100% Whey Protein GNC ProPerformance from GNC,Pittsburgh, PA. **Mineral composition of AWS was ½ strength MK7-1 mediumdescribed in Table 1A but without glycerol.

Example 15: Growth of Strain MK7 Biomats on Acid Whey

Strain MK7 biomats are grown in 6 days using acid whey as the primarycarbon and nutrient source. Biomats are produced in sterilized 12.7×12.7cm (0.016 m²) polypropylene trays using 125 mL of raw acid whey that hasbeen subjected to a variety of treatments. The treatments are conductedto evaluate growth rates and biomass productivity after adjusting pH,adding select nutrients and/or heating to minimize the presence ofcompeting microorganisms.

Acid whey volumes of 500 mL are added to previously sterilized 1 LErlenmeyer flasks (heated to 125° C. for 10 minutes) and the liquidmedium is subjected to the selected treatments (1-12) as outlined inTable 5. The pH is either not adjusted (average pH of acid whey is 4.8)or adjusted to pH 2.7 with concentrated HCl. Nutrient additiontreatments include: no addition, 2.5 g/L of urea as a nitrogen source,or ½ strength MK7-1 as a full suite of nutrients prepared in the acidwhey liquid as described in Example 1, minus the glycerol. Heatsterilization is conducted by boiling the acid whey liquid medium for 15minutes after the other treatments (pH and or nutrient additions) areperformed. After the media has cooled to about 23° C., a 7.5% volume(volume/volume) of inoculum in exponential growth phase is added to theflask. This inoculum is generated as described in Example 3. Aliquots of125 mL of the inoculated culture medium are added to isopropyl swabbedtrays (see Example 3 for sterilization procedure) and the trays areplaced on larger (0.25 m²) trays mounted in the tray rack system asdescribed in Example 3. The cultures are allowed to incubate at 25±1° C.for at least 6 days.

TABLE 5 Treatment matrix used for evaluating acid whey as a nutrientmedium for growth of strain MK7 biomats. Nutrient Heat Treatment pHAddition Sterilization 1 not adjusted no no 2 not adjusted no yes 3 notadjusted Urea no 4 not adjusted Urea yes 5 not adjusted 1/2 MK7-1 no 6not adjusted 1/2 MK7-1 yes 7 2.7 no no 8 2.7 no yes 9 2.7 Urea no 10 2.7Urea yes 11 2.7 1/2 MK7-1 no 12 2.7 1/2 MK7-1 yes

Example 16: Growth of Strain MK7 Filamentous Fungi Biomats on AnaerobicDigestate

Dense strain MK7 biomats were produced in 7 days using anaerobicdigestate as the sole carbon and nutrient source (feedstock). Anaerobicdigestate is the lignin-rich solid residue that remains after themicrobial fermentation of lignocellulose-rich biomass (e.g. corn stover,wheat straw, cattle manure) under oxygen limited conditions. Anaerobicdigestate is considered resistant to further decomposition bymicroorganisms and for this reason is commonly used as a soil amendmentor is burned to power steam generators for production of electricity.

Moist anaerobic digestate (500 g) was added to 2 L of drinking qualitytap water forming a mixture. The native pH of this mixture was 5.5. Themixture was inoculated with 7.5% (volume:volume) of strain MK7 inoculum,prepared as described in Example 3. 200 ml of the resulting mixture wasadded to square 12.7×12.7 cm trays. Consolidated biomats formed on thesurface and were harvested after 7 days of incubation at roomtemperature (˜23° C.). The biomats had an average thickness of 2.6 mmand had an average dry weight of 0.62 g (n=3, standard deviation=0.03 g)and a corresponding density of 0.015 g/cm³. The average conversionefficiency was 2.3%.

To enhance rates of anaerobic digestate conversion to microbial biomass,an additional experiment was conducted by supplementing the anaerobicdigestate with Barley Medium, effectively using an augmented growthmedium. The Barley Medium was used to stimulate growth and induce insitu enzyme production by strain MK7 for further degradation andconversion of anaerobic digestate to fungal biomass. The augmentedBarley Medium was prepared by combining 1 L of tap water, 50 g of barleyflower, 1 g yeast extract, 0.1 mL glucoamylase (Distillate VHP, Dupont),0.1 mL alpha-amylase (SPEZYME ALPHA, 13,775 AAU/g, Dupont) and 0.1 mLbeta-gluconase (Optimash TBG, Dupont). The mixture was heated to 65 Cand stirred for 15 minutes while at 65 C. Afterward, the mixture wasboiled for 15 minutes to deactivate the enzymes. The mixture was thencooled to room temperature.

The protocol described above for the anaerobic digestate experiment wasrepeated with exception that the tap water was substituted withaugmented Barley Medium and the total volumes of each component usedwere reduced by one half. The average conversion of anaerobic digestateto biomats was 6±2% after subtraction of biomass generated in thecontrol treatment where no anaerobic digestate was added.

Example 17: Growth of Other Filamentous Fungi in Tray Reactors

The growth of Rhizopus oligosporus and Fusarium venenatum were evaluatedusing the surface fermentation techniques described in Examples 1, 2,and 3 for strain MK7.

Rhizopus oligosporus is extensively used for Tempeh (human food)production around the world. Rhizopus oligosporus strain ATCC 22595 wasobtained from ATCC on Oct. 1, 2015. The pure culture sample of R.oligosporus obtained from ATCC was placed on MK7-1 agar medium in Petriplates as described in Example 2.

Fusarium venenatum used in Quorn™ food production was culled from aQuorn Chik'n Nuggets package (UPC code: 33735-00006) purchased on Jan.16, 2016 from Albertson's supermarket in Bozeman, Mont. To isolate F.venenatum, a sample (˜0.25 cm²) of Quorn™ Chik'n Nuggets containing F.venenatum was placed in 100 mL of sterile pH 5, MK7-1 medium in a 250 mlbaffled shaker flask. The medium and flask were sterilized by boilingthe medium in the flask for 20 minutes. The medium was allowed to coolto 23 C prior to addition of the Quorn™ sample. After 3 days ofincubation at 23±1 C while rotating at 200 rpm, 1 mL of culture wasremoved and used to inoculate another identical flask and medium. After3 more days of incubation in the same manner, a 50 μL aliquot of theculture was removed and plated on sterile MK7-1 agar medium (pH 4.8) ina sterile Petri plate.

The mycelial mats that developed on the R. oligosporus and F. venenatumplates were used to inoculate 350 mL of MK7-1 medium in sterile 1 Lbaffled shaker flasks as described in Example 3. After 5 days of growthin the shaker flasks as described in Example 3, the cultures were usedas inoculum for tray reactors. Desirable inoculum for the tray reactorsconsists of microbiologically pure cultures with cellular densitygreater than 6 g/L that are in the late exponential phase (see Example3). As described in Example 3, two trays containing 1.5 L of inoculatedMK7-1 medium were prepared for each of the two organisms. The pH of themedia was adjusted to 4.1 for Rhizopus and 5.0 for Fusarium. Images ofthe resultant cultures and mats are shown in FIGS. 12 and 13.

Example 18: Comparison of Filamentous Fungi Biomat Produced bySolid-State Fermentation (SSF) Compared to Biomass Produced by SolidSubstrate Surface Fermentation (SSSF) SSF Procedure

Solid-state fermentation (SSF) as referred to herein means the microbialfermentation process that occurs on solids at low water contents,typically below 50%.

MK7 SSF inoculum was prepared by the addition of 20 g glucose to 1 LMandels and Reese medium in a 2 L glass vessel and autoclaved for 45minutes at 121° C. at pH 4.5. 100 ml of the resulting medium was addedto a 250 ml Erlenmeyer flask and inoculated with 0.25 g of strain MK7from −80° C. glycerol stock. The culture was incubated at 30° C., 180rpm for 14 days before use as inoculum for SSF.

An example of the SSF process is that which was used for the productionof biomats in WO 2016/004380 and US 2016/0002680. Specifically, 100grams of wheat straw was size reduced in a commercial blender and placedin a 2 liter glass bottle. 300 ml of Mandels and Reese medium and 3 mlof concentrated H₂SO₄ was added. The resulting slurry was autoclaved for45 minutes at 121° C. The pH of the autoclaved slurry was adjusted withNaOH to 3.0, allowed to cool to room temperature, and then transferredequally between four 250 ml Erlenmeyer flasks. 10 ml of strain MK7inoculum was added and the flasks shaken at 30° C., 180 rpm for 4 daysfollowed by transfer of the contents of all flasks to a single 9×9 inchPyrex® dish. The resulting culture was covered with Saran® wrap andincubated at 30° C. for 7 days before harvesting.

SSSF Procedure

The SSSF inoculum is described in Example 3 and procedures are describedin Examples below (i.e. conversion of sugar beet pulp and otherlignocellulosic materials that float on top of a liquid layer.Specifically, as referred to herein SSSF means fermentation that occurswhen a solid substrate is submerged under the surface of a liquid, suchthat a filamentous fungi biomat grows on the surface of the liquid usingcarbon and nutrients derived from the submerged solid. Filamentous fungibiomats produced are cohesive, dense, and free of feedstock (FIG. 14C).

The biomass production and the resulting biomats produced differconsiderably between the SSSF and SSF procedures. Medium components,ionic strength, osmotic pressure, feedstock concentration, inoculumquality, cultivation time (Tables 6 and 7) are all important parameterdifferences between SSSF and SSF methodologies. These processdifferences result in vastly different biomass properties (e.g. density,formation of a consolidated mat, microbial purity, filament length,filament organization, etc.).

Biomass produced by SSSF results in the production of filamentous fungibiomats that float on top of a liquid layer and that are physicallyseparate from a solid feedstock layer. The resulting filamentous fungibiomat is an essentially pure fungal biomass organized into a cohesiveand dense mat. The mat has a high tensile strength and is comprised oflong filaments with average lengths spanning from millimeters tomultiple centimeters (as shown in FIGS. 1C, 11 and 12). The filamentsare predominantly organized parallel to the surface of the biomat andare connected by highly dense clumps of fungi. The surface of the biomatmay or may not exhibit aerial hyphae that are oriented perpendicular tothe surface of the biomat. The filamentous fungi biomat is easilyremoved from the growth environment as it is physically separate fromthe liquid or solid substrate, which enables rapid and easy harvestingof relatively pure filamentous fungi biomats.

In contrast, biomass grown by SSF produces biomass heterogeneouslyintegrated with the solid substrate in a random configuration. Thebiomass filaments produced are generally less than 100 μm in length(FIG. 14B). Further, the biomass produced by SSF is not cohesive andsuffers from low tensile strength such that it cannot be picked up in asingle unit (see FIG. 14A). The resulting biomass/solid substratemixture tends to be of low density, particularly when compared tofilamentous fungi biomats produced by SSSF.

TABLE 6 The following describes key differences in SSF and SSSFmethodologies: SSF SSSF Density (g dry weight strain <5 120-180 MK7biomass/kg of medium:substrate mixture Tensile Strength Not measurableWet biomass: 0.05-0.24 kg/ com width, average ~0.009 kg/com width. Drybiomass (no subsequent processing): 2-6 kg/com width, average ~3 kg/comwidth Osmotic pressure of medium 3.4 18.6 (atm) Ionic strength of medium0.077 0.368 (molar) Cell type used for inoculum Stationary phasefilamentous Late exponential phase cells (i.e. cells >100 μm inplanktonic cells (i.e. cells length) <20 μm in length) Lignocellulose inmedium >25 2.5-25 (%) Average filament length 0.001-0.02 cm 0.05-2 cmFilament orientation Random Parallel Final composition (%) Less than 5%fungi Greater than 95% fungi Final biomat consistency Fragile, notcohesive, Robust, cohesive, heterogeneous homogeneous

Example 19: SSF and SSSF of Sugar Beet Pulp by Strain MK7

Strain MK7 biomass was produced using SSF and SSSF methods with sugarbeet pulp as the primary carbon source. Beet pulp is the vegetableportion of the sugar beet that remains after the sugar has been removedfrom the beet pulp at the processing plant. Sugar beet pulp was obtainedfrom the Western Sugar Cooperative production plant in Billings, Mont.,and was comprised of approximately 24% dry matter, 9.1% crude protein,0.6% crude fat, 23.1% crude fiber, 4% ash, and 0.56% calcium.

For the SSF experiment, 50 g of dry beet pulp was mixed with 250 mlwater. The mixture was autoclaved for 20 minutes to ensure sterility.After cooling to room temperature (˜23° C.), the mixture was inoculatedwith 250 mg of moist strain MK7 biomat that was grown as a biomat on thesurface of a corn stover/water mixture. The strain MK7 biomat was mixedinto the beet pulp mixture with a sterilized spatula and the resultinginoculated mixture was allowed to incubate at room temperature for 4, 5,and 7 days.

For the SSSF experiments, beet pulp was added at a concentration of 7%to ½ strength MK7-1 medium (pulp weight:liquid volume) to create 300 mlof medium. The pH of the medium was adjusted to 4.8 by the addition of asuitable amount of HCl, followed by boiling for 20 minutes. Aftercooling to room temperature, approximately 100 mg of MK7 biomat that wasgrown as a filamentous fungi biomat on the surface of a cornstover/water mixture as described in Example C was mixed into the mediumwith a sterilized spatula. The pH was re-adjusted to 4.8 by the additionof a suitable amount of HCl and 100 ml aliquots of the inoculated pulpmedium was then added to sterilized 0.023 m² Pyrex® glass trays prior toplacing the trays in a tray rack system. The inoculated mixture wasincubated at 23°±1° C., resulting in flexible, dense biomats that wereabout 2.9, 3.4, and 4.1 mm thick after 4, 5, and 7 days, respectively.The biomats were harvested and dried at 50° C. for 48 h and the dryweights were 1.85 g (4 days), 2.25 g (5 days), and 2.85 g (7 days). Theconversion of beet pulp to dry biomat was 26.4%, 32.1%, and 40.7% forthe 4, 5, and 7 day mats, respectively. Biomat densities based on dryweight of the most biomat volume were 0.028, 0.029, and 0.030 g/cm³ forthe 4, 5, and 7 day mats, respectively.

Significantly different biomass forms resulted from growth using SSFversus SSSF. SSF produced biomass structures that were intimate mixturesof both biomass and substrate. These mixtures were comprised of lowdensity fungal biomass intertwined around and within fragments of thebeet pulp substrate. These intimate mixtures were primarily comprised ofthe substrate interspersed with a small amount of fungal biomass.Separation of the biomass from the substrate was not done as it wouldrequire significant additional process and would be technicallydifficult to accomplish.

In contrast, SSSF resulted in filamentous fungi biomats that werephysically separate and distinct from the beet pulp substrate, allowingdirect and straightforward harvesting of the biomat. Further, theresulting filamentous fungi biomats were dense, essentially pure andcomprised of long aligned filaments.

Example 20: Growth of Strain MK7 Filamentous Fungi Biomats on Carrot andBroccoli Waste

Dense strain MK7 biomats were produced in 6 days using homogenizedbroccoli or homogenized carrots as feedstocks. The broccoli and carrotswere purchased from Costco Wholesale in Bozeman, Mont. 100 grams of eachfeedstock was individually homogenized in a commercial food processorusing a metal blade at high speed for 5 minutes and placed in 2-literbeakers with 9000 ml of tap water. Medium salts were added as follows toform a mixture:

g/L NH₄NO₃ 5.25 Urea 1.75 KH₂PO₄ 5.0 CaCl² 1.0 MgSO₄•7H₂O 1.0Micronutrients mg/L FeSO₄•7H₂O 2.50 Zn SO₄•7H₂O 1.10 MnCl₂•4H₂O 0.25CoCl₂•6H₂O 0.08 CuSO₄•5H₂O 0.08 (NH₄)₆Mo₇O₂₄•4H₂O 0.06 H₃BO₃ 0.06 EDTA,free acid 19.63

The pH of the mixture was adjusted to 3.5 by adding 1.3 ml concentratedHCl. The medium was covered with aluminum foil and then boiled for 30minutes. After cooling to room temperature (˜23° C.), 50 ml (7.5%volume:volume) of inoculum prepared as described in Example 3 was addedto the medium for each feedstock and stirred until a homogenous mixtureformed. 250 ml of mixture was poured into four separate 5×7 inch (0.02m²) trays and covered with Saran® wrap. The trays were cultivated for 7days at 23°±1° C. before harvesting. The resulting biomass was aflexible, dense filamentous fungi biomat free of the remaining broccolior carrot feedstock. That is, a filamentous fungi biomat was producedthat did not contain feedstock residue and was comprised of essentiallypure MK7 biomass. The mean pH value of the residual liquid afterharvesting was 6.2. The average thickness of the moist biomats was 3±1mm. Filamentous fungi biomats were dried at 50° C. for 72 h and theaverage dry weights ±standard deviations were 1.7±0.2 g for broccoli and1.7±0.2 g for carrots. The average conversion of broccoli to dry weightwas 52±5 g strain MK7 dray weight/100 g dry weight broccoli. The averageconversion of carrots to dry weight is 55±7 g strain MK7 dry weight/100g dry weight carrots.

Example 21: Strain MK7 Cultivation on Municipal Organic Waste Surrogate(Grass Clippings and Leaves as a Function of Pretreatment)

The impact of acid and base pretreatments on municipal organic waste wasevaluated as a function of the percentage conversion of feedstock tofilamentous fungi biomat. Kentucky bluegrass clippings and ash treeleaves were separately dried at 60° C. until water content was less than8%. Each feedstock was ground in a commercial blender into a finepowder.

HCl Acid Pretreatments

-   -   3 replicates of 10 g of grass/leaves (50:50 by dry weight) in        100 ml tap water at pH 2.5 (adjusted with 33% HCl) were        pretreated by boiling 10 minutes.    -   3 replicates of 10 g of grass/leaves (50:50 by dry weight) in        100 ml tap water at pH 2.5 (adjusted with 33% HCl) with 10 mM        MnSO₄ were pretreated by boiling 10 minutes.    -   3 replicates of 10 g of grass/leaves (50:50 by dry weight) in        100 ml MK7-1 medium at pH 2.5 (adjusted with 33% HCl) were        pretreated by boiling 10 minutes.    -   3 replicates of 10 g of grass/leaves (50:50 by dry weight) in        100 ml MK7-1 medium at pH 2.5 (adjusted with 33% HCl) with 10 mM        MnSO₄ were pretreated by boiling 10 minutes.

NaOH Base Pretreatments

-   -   3 replicates of 10 g of grass/leaves (50:50 by dry weight) in        100 ml tap water at pH 10.75 (adjusted with 1% NaOH) were        pretreated by boiling 10 minutes. Final pH 2.5 was adjusted with        HCl.    -   3 replicates of 10 g of grass/leaves (50:50 by dry weight) in        100 ml tap water at pH 10.75 (adjusted with 1% NaOH) with 10 mM        MnSO₄ were pretreated by boiling 10 minutes. Final pH 2.5 was        adjusted with HCl.    -   3 replicates of 10 g of grass/leaves (50:50 by dry weight) in        100 ml MK7-1 medium at pH 10.75 (adjusted with 1% NaOH) were        pretreated by boiling 10 minutes. Final pH 2.5 was adjusted with        HCl.    -   3 replicates of 10 g of grass/leaves (50:50 by dry weight) in        100 ml MK7-1 medium at pH 10.75 (adjusted with 1% NaOH) with 10        mM MnSO₄ were pretreated by boiling 10 minutes. Final pH 2.5 was        adjusted with HCl.

Control Pretreatments

-   -   3 replicates of 10 g of grass/leaves (50:50 by dry weight) in        100 ml tap water. Final pH 5.5.    -   3 replicates of 10 g of grass/leaves (50:50 by dry weight) in        100 ml tap water, 10 mM MnSO₄. Final pH 5.5.    -   3 replicates of 10 g of grass/leaves (50:50 by dry weight) in        100 ml MK7-1 medium. Final pH 5.5.    -   3 replicates of 10 g of grass/leaves (50:50 by dry weight) in        100 ml MK7-1 medium, 10 mM MnSO₄. Final pH 5.5.

The samples were placed in 12.7×12.7 cm trays, covered, and thenincubated for 7 days. Results are shown in FIG. 15. In each case, theapplication of a pretreatment increased the resulting conversionpercentage. That is, a greater amount of conversion of the feedstock toresulting filamentous fungi biomat was achieved by the application of anacid or base pretreatment and by the addition of manganese.

Example 22: Growth of Strain MK7 Biomats on Starch

Dense strain MK & filamentous fungi biomats were produced in as littleas 4 days using starch as a carbon and nutrient source (feedstock). Thestarch used in these specific experiments was 100% Argo Corn Starchmanufactured by Argo Food Companies, Inc (Memphis, Tenn.) and purchasedfrom Albertson's supermarket in Bozeman, MH.

Three batches of starch media were prepared by adding 6%, 8%, and 10%dry starch powder to 6 L volumes of drinking quality tap water in steel10 L pots. This mixture was supplemented with MK7-1 salts and boiled for10 minutes followed by cooling to room temperature (˜23° C.). Heatingthe mixture resulted in coalesced clumps of starch that were thenphysically broken into smaller clumps. The pH of the mixture wasadjusted to 2.7 and inoculated with 7.5% (volume:volume) of MK7 inoculumprepared as described in Example 3.

Aliquots of 1.5 L inoculated media were added to four sanitizedpolypropylene 0.25 m² trays, placed in a tray rack system, and incubatedat 23°±1° C. Dense filamentous fungi biomats were observed after just 2days of growth and the biomats harvested after 6 days. The mean pH valueof the residual liquid remaining in the trays after harvesting was 6.05,6.11, and 5.88 for the 6%, 8%, and 10% treatments, respectively. Themean thickness of the biomats were 2.9, 3.1, and 3.3 mm for the threetreatments, respectively. Filamentous fungi biomats were dried at 50° C.for 72 h and the average dry weights ±standard deviations were 29.0±1.3,34.4±1.5, and 38.2±1.9 g for the four replicate trays containing the 6%,8%, and 10% starch, respectively. This is equivalent to a conversionpercentage of 32, 29, and 25% starch to filamentous fungi biomats dryweight. Average densities on a dry weight basis for the moistfilamentous fungi biomats were 0.04, 0.04, and 0.05 g/cm² for the threetreatments, respectively.

Example 23: Growth of Strain MK7 Filamentous Fungi Biomats on PotatoProcessing Waste Streams

Dense strain MK7 filamentous fungi biomats were produced in 7 days usingpotato processing waste as carbon and nutrient source (feedstock).Potato processing waste is commonly produced during the processing ofpotatoes and includes waste streams from washing, peeling, and cuttingoperations (i.e. French fries, potato cubes, flakes, and the like).Potato processing waste streams also include discharge piles comprisedof multiple potato processing waste streams pilin in a heap and exposedto the natural environment with no coverings. In this example, potatoprocessing waste stream comprised of processing waste of multiplevarieties of potatoes was obtained from Bauch Farms in Whitehall, Mont.on Sep. 21, 2016, and used within 48 hours as a carbon and nutrientsource to grow strain MK7 biomats.

Potato shorts are those pieces of potato that remain after French friesare cut from a full potato. Potato shorts vary in size and dimensionsspanning, as a non-limiting example, from thin slivers to pieces thatare 6 inches long by 0.5 inches thick or more. Fresh discards, in themajority of cases, describes those pieces of potato that are removedfrom a potato due to damage, bruising, or the like. In some cases, wholepotatoes are included as discard samples. Peels are predominantly skinsremoved from potatoes.

Potato shorts, discards and skins were processed by a food processor toa homogenous consistency (Farbarware Model 103742 food processor set onhigh) in approximately 500 ml volume batches for 1 minute. Foodprocessed samples are termed blendate for the purposes of description inthis example.

Blended potato shorts and fresh discards were added to two 15 Lepoxy-coated steel cooking pots at a ratio of 10% wet weight blendate toa volume of MK7-1 medium at a ratio of 500 g blendate to 4.5 L liquidMK7-1 medium, producing a mixture. The pH of the mixture was adjusted to2.45 with concentrated HCl. Strain MK7 inoculum, prepared as describedin Example 3, was added at a ratio of 7.5% volume:volume (i.e. 375 mlinoculum to 4625 ml mixture. Aliquots of 1.5 L inoculated suspensionwere added to individual 0.25 m² sterilized polypropylene trays intriplicate and placed in a tray rack system. The cultures were incubatedat 23°±1° C., resulting n flexible, dense biomats harvested after 7days. The mean pH value of the residual liquid remaining in the traysafter harvest was 7.1 for the shorts and 6.9 for fresh discardstreatments. Harvested biomats were rinsed in 7 L tap water with gentleagitation for 10 minutes and dried at 50° C. for 72 h.

The average thickness of the moist biomats was 3.8±0.9 mm for the shortsand 3.9±1.0 mm for the discards. The average dry weights ±standarddeviations of the biomass in each tray were 33.6±0.6 g from the shortsand 40.2±2.7 g for the fresh discards. The average density of thebiomats based on dry weight was 0.035 g/cm³ for the shorts and 0.041g/cm³ for the discards. The average conversion of total solids in theoriginal potato by-products to dry biomat was 36% for the shorts and 43%for the fresh discards. Near 50% conversion would be considered 100%conversion efficiency of the carbon given the fact that about 50% of thecarbon used by strain MK7 is released to the atmosphere as carbondioxide.

Blended potato peels were pretreated prior to growth experiments toincrease access of potato peel nutrients to strain MK7. Blended potatopeels (175 g) were added to each of nine 12.7×17.8 cm (0.023 cm²) Pyrex®glass trays to create an experimental matrix of three treatments withthree replicates each. Treatment 1 received 50 ml drinking quality tapwater. Treatment 2 received a 45 ml aliquot of strain MK7 hydrolysatecontaining a suite of strain MK7 hydrolytic enzymes excreted by strainMK7 when grown on corn stover. Treatment 3 received 50 ml tap water anda suite of commercial enzymes comprised of 0.05 g cellulase Y-C (MPBiomedicals, Cat#320951, Lot# M4156), 2.5 ml glucoamylase (DistillateVHP, Dupont), 2.5 ml alpha-amylase (APEZYME ALPHA, 13,775 AAU/g, Dupont)and 2.5 ml beta-gluconase (Optimash TBG, Dupont). Treatment 3 trays wereincubated at 50° C. for 30 minutes to stimulate enzymatic hydrolysisfollowed by boiling for 5 minutes to inactivate the enzymes. The pH ofthe treatments was adjusted to 3.0 using concentrated HCl and all trayswere inoculated with 10 ml of strain MK7 prepared as described inExample 2. After 7 days, the biomats were removed from the surface ofthe liquid, rinsed in tap water for 10 seconds and dried at 60° C. for48 h. Conversion of potato peel dry weight to dry biomats were: Control(H₂O only) mean=5.9% (5.5%, 6.5%, and 5.8%); Strain MK7 enzymes=9.0%(7.2%, 10.1%, and 9.8%); and Commercial enzymes=9.9% (10.2%, 8.4%, and11%).

Example 24: Nutritional Analysis Biomass Produced by SSF Versus BiomatsProduced by SSSF

Nutritional analysis was performed by Eurofins comparing the biomatsresulting from SSF versus SSSF methodologies. SSF samples were obtainedfrom strain MK7 cultivated on corn stover pretreated with ammonia fiberexpansion (AFEX) by Michigan Biotechnology Institute. 150 g of AFEX wereadded to 500 ml of tap water and autoclaved at 121° C. after adjustingthe pH to 3.5 with concentrated HCL. Resulting mixture was inoculatedwith 25 ml of strain MK7 inoculum according to Example 18. Slurry wastransferred to a 23×23 cm Pyrex® glass tray and incubated at roomtemperature for 11 days. Integrated strain MK7 biomass and corn stoverwas harvested and dried at 60° C. for 48 hours. Samples were analyzedfor total protein, total fiber, total carbohydrates, ash, and total fatsby Eurofins USA (Des Moines, Iowa).

SSSF samples were obtained from mats produced on 5% AFEX corn stover. 50g of AFEX corn stover was added to 1 L of tap water and autoclaved at121° C. after adjusting the pH to 3.5 with concentrated HCl. Theresulting mixture was inoculated with 50 ml of strain MK7 inoculumprepared as described in Example 3. Slurry was transferred to two 23×23cm Pyrex® glass tray and incubated at room temperature for 11 days. Matswere harvested and rinsed in tap water for 30 seconds followed by dryingat 60° C. for 24 hours. Samples were analyzed for total protein, totalfiber, total carbohydrates, ash and total fats by Eurofins USA (DesMoines Iowa).

TABLE 7 Eurofins Analysis SSF (%) SSSF (%) Total protein 2.56 51.10Total fat 0.60 12.00 Total fiber 80.30 23.30 Total sugars 2.10 <0.35Total ash 15 12.40

Example 25: Amino Acid Profile of the Filamentous Acidophilic MK7 FungalStrain

The filamentous acidophilic MK7 fungal strain biomat was produced intray reactors using the method described in Examples 2 and 3 in theMK7-1 medium. The filamentous biomass from 6 trays was combined prior todrying at 60° C. for 45 minutes and 50° C. for 72 hours. 400 g of thisfilamentous biomass was sent to Eurofins Scientific Inc. NutritionalAnalysis Center in Des Moines, Iowa, for nutritional analysis. Aminoacids were analyzed using the internationally recognized methodspublished in the Association of Official Agricultural Chemists (AOAC)Official Methods of Analysis as follows: AOAC 988.15 for Tryptophan,AOAC 994.12 mod. for Cystine and Methionine, AOAC 982.30 mod. forAlanine, Arginine, Aspartic Acid, Glutamic Acid, Glycine, Histidine,Isoleucine, Leucine, Phenylalanine, Proline, Serine, Threonine, TotalLysine, Tyrosine and Valine. The amino acid composition of thefilamentous acidophilic MK7 fungal strain sample reported by Eurofins iscompared with the amino acid composition of Fusarium venenatum used forfish food (Alriksson, B. et al. (2014) Fish feed from wood. CelluloseChemistry and Technology 48:9-10 (2014), Quorn (Nutritional Profile ofQuorn Mycoprotein, 2009), egg albumin (Food and Agriculture Organizationof the United Nations. The Amino Acid Content of Foods and BiologicalData on Proteins, Nutritional Study #24. Rome (1970). UNIPUB, Inc.,4611-F Assembly Drive, Lanham, Md. 20706) and Rhizopus oligosporus(Graham, D. C., Steinkraus, K. H. & Hackler, L. R. (1976) Factorsaffecting production of mold mycelium and protein in synthetic media.Appl Environ Microbiol 32:381-387) in Table 8. The total protein contentwas measured as 41.5% of a 4.5% moisture content biomass. Notably, thefilamentous acidophilic MK7 fungal strain was shown to have a higherconcentration of essential amino acids compared to all of the four otherprotein sources, making the filamentous acidophilic MK7 fungal strain ahighly desirable source of protein for food and feeds.

TABLE 8 Amino acid concentration as a percent of total amino acids areprovided for the filamentous acidophilic MK7 fungal strain and four highprotein food/feed sources. Essential amino acids are denoted with anasterisk. Fusarium venenatum Rhizopus Strain for Egg oligosporus MK7fish food Quorn albumin (Tempeh) *Tryptophan 1.48% 0.94% 1.24% 1.18%0.75% Cystine 1.04% 1.88% *Methionine 1.65% 1.51% 1.59% 3.01% 0.58%Alanine 16.38% 5.49% Arginine 5.39% 4.72% 4.54% Aspartic 9.17% 6.09%Glutamic 10.72% 10.89% Glycine 5.06% 2.89% *Histidine 2.12% 2.69% 1.67%*Isoleucine 4.48% 3.96% 3.93% 5.00% *Leucine 6.84% 5.85% 6.55% 6.80%*Phenylalanine 3.57% 3.72% 4.94% Proline 4.35% 2.92% Serine 4.45% 6.07%*Threonine 5.49% 3.77% 4.21% 3.41% 3.05% *Lysine 7.25% 5.66% 6.28% 4.64%4.28% Tyrosine 2.70% 3.21% *Valine 7.85% 4.72% 4.14% 6.02%

Example 26: Production of C18-Rich Lipids by the Filamentous AcidophilicMK7 Fungal Strain from Food Grade Glycerol

Medium Preparation:

4.5 liters of MK7-1 medium was prepared with 125 g/L glycerol (TheChemistry Store—Kosher Food Grade Glycerol >99.7%, ASIN: B00KN1LRWQ,available on the internet) (562.5 grams) with NH₄NO₃ and Urea nitrogenconcentrations altered to a C:N ratio of 40:1 (mols carbon in Csource:mols N in nitrogen compounds).

TABLE 9 Composition of MK7-1 medium modified to provide a C:N ratio of40:1 and 12.5% glycerol concentration. Micronutrients were supplied byadding 2 mL/L of a 500x stock solution described in Table 1 (Example 1).Total Volume 4.5 NH₄NO₃ (g) 10.8 Urea (g) 3.7 CaCl₂ 9.0 MgSO₄—7H₂O (g)9.0 KH₂PO₄ (g) 45.0 Micronutrients (mL) 9.0 Glycerol (g) 563 Glycerol(L) 0.446 C:N ratio 40:1 pH 2.7 Deionized H2O (L) 4.05 HCl (mL) 5.85

The mixture pH was adjusted to 2.7 and heat sterilized by boiling for 30minutes in a 2 liter Erlenmeyer flask with top of flask covered withaluminum foil. The mixture was cooled for 2 hours to 25° C.

Inoculation:

Inoculum (15 g/L planktonic cells as dry weight in exponential growthphase (see Example 3) was added to the cooled flask at a final dry weighconcentration of 1 g/L. The flask was thoroughly mixed for evendistribution of inoculum. Planktonic state cells are critical for matformation and it is desired that cell clustering (i.e. biofilm greaterthan 1 mm) be minimized. Ideally, cell clusters greater than 2.5 mmshould be filtered from the inoculant prior to distribution.

Incubation and Harvesting:

The mixture with inoculum was evenly distributed in three 0.25 m² traysat a volume of 1.5 liters/tray or 6 liters per square meter andincubated at 25 C, 90-100% humidity for 8 days. A consolidated biomatbiomass is produced at cell densities above 30 g/L and biomass is ableto be harvested as one cohesive mat. In one embodiment the mat is simplyrolled off the tray (FIG. 6). The mat is rinsed for 30 seconds usingrunning water and allowed to drip dry for 5-10 minutes. Squeezing of themat was avoided as protein and other fungal nutrients are lost throughexcessive water removal. The filamentous biomass after drip drying had awet weight of 410 grams (or 1,620 grams/m²). Moisture content wasmeasured at 82% (i.e. dry weight of 18%) corresponding to dry weight of73.8 g/tray or 295 g/m². Dry weight filamentous biomass of 18% comparesfavorably to processing of other fungal biomass grown in submergedcultures with typical dry weight of 1.5%. In contrast, state of the artprocesses utilize centrifuges (an energy and capital intensive process)to achieve desired fungal biomass density. The process described hereinrequires far less processing, equipment and energy input compared tothese more expensive methods.

Lipid Analyses:

Estimates of total lipids were done by the UV-Vis microscopy with NileRed staining (Cooksey et al., 1987; FIG. 16), which estimated totallipids at 40-50%. Quantification of total intracellular lipids wasdetermined using direct transesterification coupled with GC-MS analysisas described in Lohman et al. (2013) and was found to be 39%. Thiscorresponds to lipid production of 115 g lipid/m² in 8 days (14g/m²/day) or 0.39 g/liter/hour average production rate. These rates aremuch faster than those found in submerged cultures with the filamentousacidophilic MK7 fungal strain with 8% glycerol (0.245 g/L/hr) and verycompetitive to other organisms found in the literature including yeastand algae. Furthermore, the filamentous acidophilic MK7 fungal strainproduces lipids at these competitive rates at very high glycerolconcentrations not tolerable by most organisms and is the only organism(to our knowledge) that can do this at acidic pH ranges, which hassignificant advantages for limiting contamination. Furthermore, thelipid coefficient (g lipid/g substrate) is highly competitive to otherstrains at 0.21 g lipid/g glycerol (see attached table). Increased lipidproduction rates and cell densities of 180 g/L have direct implicationsto transform the production of microbial oils by a wide variety ofmicroorganism currently being developed or in commercial use.

The filamentous acidophilic MK7 fungal strain lipid profiles areremarkably consistent among different types of treatments (i.e. pH,temperature, growth substrate, cultivation duration, moisture content)and are dominated by C16:0 and C18:0, C18:1 and C18:2 triacylglycerides(>95% of total lipids; Table 10 below; FIG. 17). Fatty acid profilesalso show a number of high value products including the omega-7 vaccenicacid (Methyl 11-octadecenoate), omega-7 palmitoleic acid (methylhexadec-9-enoate; trade name Provinal™) and tetracosanoic acid, methylester. These are rare fatty acids not typically found in vegetable oilsand may produce significantly more revenue per ton of feedstock thanbiodiesel alone.

TABLE 10 Identities, concentrations of fatty acids found in strain MK7biomass cultivated with 12.5% glycerol for 8 days 30° C.; and C:N ratioof 40:1 SB (n = 3) EuroFin % of % of FAME FAME Profile Profile STD C10:0(Capric acid) 0.3% 0.1% 0.01% C11:0 (Undecanoic acid) 0.3% 0.0% 0.01%C12:0 (Lauric Acid) 0.3% 0.0% 0.00% C14:0 (Myristic acid) 0.5% 0.4%0.00% C14:1 (Myristoleic acid) 0.3% 0.0% 0.00% C15:0 (Pentadecanoicacid) 0.3% 0.3% 0.03% C16:0 (Palmitic Acid) 15.8% 21.2% 0.45% C16:1Omega 7 1.0% 0.8% 0.05% C17:0 (Margaric Acid) 0.3% 0.1% 0.00% C18:0(Stearic Acid) 4.8% 14.7% 0.39% C18:1 (Oleic Acid/Isomers) 23.0% 31.9%0.14% C18:2 Omega 6 (Linoleic Acid) 42.8% 26.8% 0.60% C18:2 (Isomers)1.0% 0.2% 0.00% C18:3 (Linolenic Acid/ 2.3% 0.9% 0.07% Isomers) C20:0(Arachidic Acid) 0.3% 0.7% 0.02% C20:1 (Gadoleic Acid/ 0.3% 0.1% 0.01%Isomers) C21:5 Omega 3 0.3% 0.0% 0.01% (Heneicosapentaenoic Acid) C22:0(Behenic Acid) 0.3% 0.5% 0.03% C22:1 (Erucic Acid/Isomers) 0.3% 0.0%0.00% C24:0 (Lignoceric Acid) 0.7% 0.7% 0.00% C24:1 (Nervonic Acid/ 0.3%0.0% 0.00% Isomers) Total %: 94.7% 99.4%

Example 27: Toxicity Analyses of Strain MK7

Five samples of strain MK7 grown under different conditions were assayedfor the presence of mycotoxins. Sample 1 biomass was produced in a 10 Lbioreactor under the same conditions used to generate inoculum asdescribed in Example 1, with exception that the C:N ratio was 30:1. Thebiomass sample was collected by filtering through a 0.2 um filter usinga vacuum filtration apparatus as described in Example 1.

Sample 2 biomat was produced in a sterilized 12.7×17.8 cm (0.02 m²)Pyrex® glass tray using 50 mL of pH 2.8 MK7-1 medium prepared asdescribed in Example 1, with exception that the media was supplementedwith 12% glycerol and 0.2% peptone (weight/volume; Peptone granulated,Fisher Scientific, Lot#143241, Somerville, N.J.). Sample 2 used the sameprocedure for sterilization as used for 0.25 m² trays related in Example3.

Sample 3 was grown in conditions identical to Sample 2, with exceptionthat the pH was adjusted to 4.5 and the media was not supplemented withpeptone.

Sample 4 was grown in conditions identical to Sample 2, with exceptionthat the medium was supplemented with 4% glycerol.

Sample 5 was grown in conditions identical to Sample 2, with exceptionthat the pH was adjusted to pH 2.2 and the media was not supplement withpeptone. Samples 2 through 5 media were inoculated with 7.5% (vol/vol)of the liquid culture used for Sample 1. Wet biomass samples werecollected after 8 days of growth and stored at −20 C prior to extractionof mycotoxins.

Mycotoxins were extracted from wet biomass using a Myco6in1+mycotoxinassay kit supplied by Vicam (Lot #100000176: Nixa, Mo.) following thestandard protocol described in the Myco6in1+ assay kit manual. Twelvedifferent mycotoxins were analyzed by LC-Q-TOF using the protocoldescribed in the Myco6in1+LC/MS/MS Instruction Manual. An Agilent 6538Q-TOF coupled to an Agilent 1290 HPLC housed at the Mass SpectrometerCore Facility at Montana State University was used for identificationand quantification of the toxins. Fumonisin B1 and Fumonisin B2 wereused as authentic standards.

Measured values for all toxins tested were below the regulatory levelsfor human consumption set by the U.S. Food and Drug Administration(Table 11). Measured levels were at least one order of magnitude lowerthan regulatory levels, with exception to Total Aflatoxins found inSample 4, which were 8.76 ng/g compared to the regulatory level of 20ng/g. However, the genes for aflatoxin production are not present instrain MK7, therefor it is expected that the source of this toxin wascontamination from peptone and other ingredients used in the medium andnot a product of MK7.

TABLE 11 Quantification of mycotoxins in biomass of strain MK7. Sample #1 2 3 4 5 Regulatory Limit Quantitation (ng/g) (wet weight) ng/gAflatoxin B1 0.03 0.04 0.02 0.49 0.02 20 * Aflatoxin B2 0.33 3.13 0.120.80 0.10 20 * Aflatoxin G1 0.02 0.02 0.04 1.91 0.04 20 * Aflatoxin G20.20 0.03 0.56 5.56 0.49 20 * Ochratoxin A 0.01 0.00 0.00 0.00 0.00 NotEstablished Deoxynivalenol 0.24 0.10 0.02 0.13 0.02 1,000 Fumonisin B10.00 0.91 0.00 4.41 0.00 2,000  

Fumonisin B2 0.00 17.40 0.04 89.58 0.02 2,000  

Nivalenol 0.12 0.08 0.04 0.37 0.04 Not Established T-2 toxin 0.07 0.000.02 0.00 0.03 Not Established HT-2 Toxin 0.13 0.11 0.04 0.14 0.05 NotEstablished Zearalenone 0.00 0.00 0.04 0.37 0.04 Not Established *TotalAflatoxins

 Total Fumonisins

The non-toxic character of MK7 culture medium and biomass was furtherverified by bioassays with Daphnia magna, a highly sensitivemacroinvertebrate commonly used for toxicity assays (EPA Publication,1987; Guilhermino et al., 2000). Live D. magna was purchased fromCarolina Biological Supply (Burlington, N.C.) and grown under theconditions described in the manual provided by the supplier. After 24hours of growth and observation, the Daphnia were used for the toxicityexperiment. Three Petri dishes were filled with 30 mL of a 30% of MK7culture (MK7 and MK7-1 medium) grown in the inoculum reactor for 6 daysas described in Example 3, and 70% water in which the D. magna wereshipped. For an experimental control, three additional Petri dishes werefilled with 30 mL of shipping water. Seven D. magna that appeared livelywere added to each of the six Petri dishes and observed daily for threedays. Death of the D. magna was defined as no visible movement after 1minute. No significant differences in survival rates were observedbetween D. magna treated with MK7 culture medium and biomass, and theexperimental controls over 3 days (average 1.2 deaths per Petri dishafter 3 days for each treatment).

The toxicity of strain MK7 biomass was also tested on Goldfish(Carassius auratus). Two identical 5.7 L fish tank, pump and filterswere purchased from Petco in Bozeman, Mont. (Aqueon model# E414W,Franklin, Wis.). The tanks were filled with 5.7 L of Poland Spring waterpurchased from the Albertson's supermarket, Bozeman Mont. Six goldfish(˜3 cm in length) were purchased from Petco (Bozeman, Mont.) and threewere placed in each one of the tanks. One of the tanks received about0.05 g of dry TetraFin Goldfish Flakes Plus (Blacksberg, Va.) fishfeeddaily (purchased from Petco, Bozeman, Mont.). The other tank receivedabout 0.0.05 g of dried strain MK7 biomass daily. The wet MK7 biomasswas obtained from one of the tray reactors produced according to theprotocol described in Example 3. MK7 biomass was prepared by removing 40g of MK7 from a tray (see Example 3) and placing the biomass in a 250 mLbeaker. The wet biomass was then microwaved using a GE microwave (ModelWES1452SS1SS) for 30 seconds. The dried biomass had a moisture contentof less than 0.5%. The biomass was then crushed with a stainless steelspatula to from small flakes that were similar in size to the TetraFinGoldfish Flakes. All fish survived and appeared to be healthy(vigorously swimming) after 60 days of feeding and showed markedenthusiasm for eating the MK7 produced biomass matt. The experiment wasterminated after 60 days.

18S rRNA and ITS region DNA sequence of the acidophilic filamentous fungal species designated as strain MK7SEQ ID NO: 1 CCGCGGGGAATACTACCTGATCCGAGGTCACATTCAGAGTTGGGGGTTTACGGCTTGGCCGCGCCGCGTACCAGTTGCGAGGGTTTTACTACTACGCAATGGAAGCTGCAGCGAGACCGCCACTAGATTTCGGGGCCGGCTTGCCGCAAGGGCTCGCCGATCCCCAACACCAAACCCGGGGGCTTGAGGGTTGAAATGACGCTCGAACAGGCATGCCCGCCAGAATACTGGCGGGCGCAATGTGCGTTCAAAGATTCGATGATTCACTGAATTCTGCAATTCACATTACTTATCGCATTTTGCTGCGTTCTTCATCGATGCCAGAACCAAGAGATCCGTTGTTGAAAGTTTTGATTTATTTATGGTTTTACTCAGAAGTTACATATAGAAACAGAGTTTAGGGGTCCTCTGGCGGGCCGTCCCGTTTTACCGGGAGCGGGCTGATCCGCCGAGGCAACAATTGGTATGTTCACAGGGGTTTGGGAGTTGTAAACTCGGTAATGATCCCTCCGCAGTTCTCACCTACGGATAGGATCATTACCGAGTTTACAACTCCCAAACCCCTGTGAACATACCCATTGTTGCCTCGGCCGGATCAGCCCGCTCCCGGTTAAAACGGGACGGCCCGCCAGAGTACCCCTAAACTCTGTTTCTATATGTAACTTCTGAGTAAAACCATAAATAAATCAAAACTTTCAACACGCATCTCTTGCTTCTGTCATCGATGAAGAACGCAGCAAAATGCGATAGTCATGTGATTGCACATTCAGTGAATCATCGATCTTGACGCACATTGCGCCTGCAGTATTCTGGCGGTCATGCCTGTTCGAGCGTCATTCAGCCCTCAGCCCTCGGTTGTGTTCGGGATCGGCGAGTCCTGCGCCAGCGACCGGATCAGTGGCGTCTGCCTGCGCCTCCATTGCGGTTAGAGTTAAGCCCTCGCCCACTTG TTTTACGCTAACTranslation elongation factor 1 alpha (Tef1) SEQ ID NO: 2ATGATCACTGGTACTTCCCAGGCCGATTGCGCCATTCTCATCATTGCCGCCGGTACTGGTGAGTTCGAGGCTGGTATCTCCAAGGATGGCCAGACCCGTGAGCACGCTCTTCTTGCCTACACCCTTGGTGTCAAGAACCTCATCGTCGCCATCAACAAGATGGACACCACCAAGTGGTCTGAGGCCCGTTACCAGGAGATCATCAAGGAGACCTCCTCCTTCATCAAGAAGGTCGGCTACAACCCCAAGGCTGTCGCTTTCGTCCCCATCTCCGGTTTCAACGGTGACAACATGCTTACCCCCTCCACCAACTGCCCCTGGTACAAGGGTTGGGAGCGTGAGATCAAGTCCGGCAAGCTCACCGGCAAGACCCTCCTCGAGGCCATTGACTCCATCGAGCCTCCCAAGCGTCCCGTTGACAAGCCCCTCCGTCTTCCCCTCCAGGATGTCTACAAGATCGGTGGTATTGGAACGGTTCCCGTCGGCCGTATTGAGACTGGTGTCATCAAGCCCGGTATGGTCGTTACCTTCGCTCCCTCCAACGTCACCACTGAAGTCAAGTCCGTCGAGATGCACCACGAGCAGCTCAGTGAGGGCCAGCCCGGTGACAACGTTGGTTTCAACGTGAAGAACGTCTCCGTCAAGGACATCCGACGTGGTAACGTCGCTGGTGACTCCAAGAACGACCCCCCCCAGGGTGCCGCTTCTTTCACCGCCCAGGTCATCGTCCTCAACCACCCCGGCCAGGTCGGTGCTGGTTACGCTCCCGTCCTCGATTGCCACACTGCCCACATTGCCTGCAAGTTCGCCGAGATCCAGGAGAAGATCGACCGCCGAACCGGTAAGGCTACTGAGGCCGCTCCCAAGTTCATCAAGTCTGGTGACTCCGCCATCGTCAAGATGGTTCCCTCCAAGCCCATGTGTGTCGAGGCTTTCACTGACTACCCTCCTCTGGGTCGTTTCGCCGTCCGTGACATGCGACAGACTGTCGCCGTCGGTGTCATCAAGGCCGTCGAGAAGTCCACCGGTGCTGCTGGCAAGGTCACCAAGTCCGCTGCCAAGGCCGCCAAGAAATAA Tubulin beta chain (Tub1): partial sequenceSEQ ID NO: 3 GTGGATCTTGAGCCCGGTCCTCAGGATGCCATCCGCGCCGGGCCCCTAGGCCAGCTTTTCCGCCCCGACAACTTCGTCGCCGGAAATGCCAGCGCCGGTAACAACTGGGCCAAGGGTCATTACACCGAAGGTGCTGAGCTCGTTGAGGAGGCCATCGATGTTGTGCGACACGAGGTTGAGAACTGTGACCATCTTCAGGGTTTCCAGCTCACCCACTCTCTCGGCGGTGGTACCGGTTCTGGTATGGGAACGCTTCTTCTGTCGAAAATCCGTGAGGAGTTTCCCGATCGCATGATGGCTACTTTTTCCGTTATGCCTTCGCCTAAGGTTTCTGATACCGTTGTCGAACCTTACAACGCCACTTTGTCATTGAACCAGCTTGTCGAGAACTCCGATGAGACCTTCTGTATCGATAACGAGGCTTTGTACGACATTTACGAGAAGACCCTGAAGATTGCTGATCCTTCTTACGCCGATCTC

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1-30. (canceled)
 31. A food product, comprising a filamentous fungalbiomat, wherein the biomat has a protein content of at least 40%,wherein the biomat comprises at least two structurally different celllayers in contact with each other, wherein the structural differencebetween the cell layers is the density within each layer, and whereinthe biomat is produced by culturing filamentous fungi to produce atleast 25 g dry wt filamentous fungi/l media.
 32. (canceled)
 33. The foodproduct of claim 31, wherein the filamentous fungi is selected from thegroup consisting of a species from a genus selected from the groupconsisting of Fusarium, Fusisporium, Pseudofusarium, Gibberella,Sporotrichella, Aspergillus, Penicillium, and Trichoderma; a specieswithin the order Mucorales; the filamentous fungal strain designated asMK7 (ATCC Accession Deposit No. PTA-10698); yeasts capable of producingfilaments; and combinations thereof.
 34. The food product of claim 31,wherein the biomat comprises amino acids selected from selected fromtryptophan, cysteine, methionine, alanine, arginine, aspartic acid,glutamic acid, glycine, histidine, isoleucine, leucine, phenylalanine,proline, serine, threonine, lysine, tyrosine and valine produced by thefilamentous fungi.
 35. The food product of claim 31, wherein the biomathas a lipid content of at least 39%.
 36. The food product of claim 31,wherein the food product is selected from group consisting of a humanfood, fish feed and animal feed.
 37. The food product according to claim31, wherein the biomat is produced by culturing filamentous fungi toproduce at least 50 g dry wt filamentous fungi/l media.
 38. The foodproduct according to claim 31, wherein the biomat is produced byculturing filamentous fungi to produce at least 120 g dry wt filamentousfungi/l media.
 39. The food product according to claim 31, wherein thebiomat is produced by culturing filamentous fungi to produce at least180 g dry wt filamentous fungi/l media.
 40. The food product accordingto claim 31, wherein the filamentous fungi are selected from the groupconsisting of strain MK7 (ATCC Accession Deposit No. PTA-10698),Fusarium species, and Rhizopus species.
 41. The food product accordingto claim 31, wherein the filamentous fungi are strain MK7 (ATCCAccession Deposit No. PTA-10698).
 42. The food product according toclaim 31, wherein the filamentous fungi are Fusarium venenatum.
 43. Thefood product according to claim 31, wherein the biomat comprises threestructurally different cell layers.